RU2673036C2 - ANTIBODY TO COMPLEMENT Bb FACTOR - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: present invention relates to antibodies and polynucleotides encoding them, which can be used to prevent, control or reduce the activity of the complement pathway. Invention is Bb factor antibody containing a heavy chain and a light chain, the specified antibody binds to factor Bb with greater affinity than with factor B, and inhibits complement-dependent hemolysis, and where the light chain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 8–11, and the heavy chain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 12–15, or the light chain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 24–27, and the heavy chain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 28–31.

EFFECT: invention relates to a pharmaceutical composition for treating or preventing an eye disease.

11 cl, 3 dwg, 4 tbl, 3 ex

Description

CROSS REFERENCE

[0001] This application claims priority according to 35 U.S.C. § 119 (e) of the provisional patent application of the United States No. 61/945613, filed February 27, 2014, and provisional patent application of the United States No. 61/947880, filed March 4, 2014, both of which are fully incorporated in this document by reference.

FIELD OF TECHNOLOGY

[0002] The present invention relates to anti-complement antibodies and compositions thereof, coding for their polynucleotides, expression vectors and host cells for producing antibodies, as well as compositions and methods for diagnosing and treating complement-mediated diseases. In particular, antibodies against factor B and against factor Bb are disclosed for use in the diagnosis and treatment of diseases associated with factor B and factor Bb, in particular age-related macular degeneration (AMD).

BACKGROUND

[0003] The complement system consists of about 50 individual proteins that function as part of the innate immune system, providing the initial stage of immune defense, the opsonization of foreign material, and tissue homeostasis. (Ricklin D., 2010, Complement: a Key system for immune surveillance and homeostasis. Nature: Immunology, 785-795) The complement system is present in all multicellular organisms and phylogenetically precedes the formation of an adaptive immune system (Zarkadis IK, 2001 Phylogenetic aspects of the complement system. Development and Comparative Immunology, 745-762.).

[0004] The activation of the complement system occurs in three main ways: classic, lectin and alternative ways. During the activation process, sequential protein-protein interactions and proteolytic activity lead to the formation of convertases C3 and C5. These convertases are responsible for the formation of complement activation cleavage products, which are effector molecules of the complement cascade, which are important for opsonization, the formation of anaphylatoxins, and the formation of a membrane-attacking complex (MAC). The latter is important for the lytic activity of the complement cascade (Ricklin D., 2010). Under normal conditions, activation of complement cascades provides protection against pathogenic bacteria, as well as clearance of affected and damaged tissue. Typically, MAC formation does not affect surrounding tissues due to the presence of cellular surface and soluble regulatory components, which include CFH, CFH-related proteins, C4BP, CD46, CD55, CD59 and complement factor I (CFI). However, when excessive activation occurs or the production of negative regulatory complement components is disrupted, both acute and chronic painful conditions are induced. Examples in which human pathologies are caused by uncontrolled complement activation include: glomerulonephritis, systemic lupus erythematosus, paroxysmal nocturnal hemoglobinuria, Alzheimer's disease, hereditary angioneurotic edema, myasthenia gravis and age-related macular degeneration (AMD) (Ricklinmune & Lambris, 2013) and inflammatory Disorders: Pthaological Mechanisms. Journal of Immunology, 3831-3838).

[0005] The complement factor B is a protein that circulates in the blood as a single chain polypeptide. After the alternative pathway is activated, factor B (about 750 ak) is cleaved by complement factor D, which leads to the formation of two polypeptides - a smaller non-catalytic Ba chain (about 230 ak; containing three domains of the complement control protein (CCP)) and a larger catalytic Bb subunit (about 510 ak; containing a protein interaction domain and a serine protease domain). Factor Bb is a serine protease that binds to C3b to form the alternative pathway C3 convertase, as well as a second protease, C5 convertase, which cleaves the C5 protein into C5a and C5b. The C5b cleavage product initiates a membrane-attack pathway, which leads to the formation of a membrane-attack complex (MAC). MAK is a transmembrane channel that leads to the osmotic lysis of the target pathogen. Thus, the cleavage of factor B and the formation of factor Bb contributes to the complement process.

[0006] Factor B is a highly regulated, highly specific serine protease. In activated form, it catalyzes a key complement activation step to initiate inflammatory responses, cell lysis, phagocytosis, and B cell stimulation (Carroll et al., Nat. Immunol. 5: 981-986 (2004)). Factor B is activated through the assembly process: it binds surface-bound C3b or its liquid phase analogue C3 (H ₂ O), after which it is cleaved by factor B into Ba fragments (residues 1-234; factor Ba, fragment Ba, complement factor Ba) and Bb (residues 235-739; factor Bb, fragment Bb, complement factor Bb). Fragment Ba dissociates from the complex, leaving the C3b-Bb complex of the C3 convertase of an alternative pathway that cleaves C3 into C3a and C3b.

[0007] Age-related macular degeneration (AMD) is the leading cause of blindness in the older generation in developed countries. Among the US population alone, the incidence of advanced forms of AMD associated with vision loss is observed in almost 2 million people. Another 7 million people with intermediate AMD are at high risk of developing advanced forms of AMD. The inclusion of the European population almost doubles the number of people affected. AMD is characterized by progressive loss of vision due to a parainflammatory process leading to progressive degeneration of neuroretin and adjacent tissues, which include retinal pigment epithelium (PES) and choriocapillaries. Most cases of clinically significant vision loss occur when neurodegenerative changes affect the region of central vision in the highly specialized region of the eye responsible for good visual acuity, the macula. The disease has a tremendous impact on the physical and mental health of a person due to loss of vision and increasing dependence on family members in carrying out everyday tasks.

[0008] The deregulation of the complement system strongly correlates with the development of AMD. First, genetic mutations in complement genes alter the risk of developing AMD in humans. In addition, AMD-related inflammation is associated with deregulation of complement activity, as evidenced by an increase in the number of complement activation products in the systemic circulation and in AMD tissue according to histopathological analysis. New discoveries have highlighted the potential pathological effects of the membrane-attacking complex in the onset of the disease (Whitmore S, et al. 2014, "Complement activation and choriocapillaris loss in early AMD: Implications for pathphysiology and therapy. Progress in Retinal and Eye Research, December 5. 2014 EPub ahead of print).

[0009] The present invention provides antibodies against factor Bb for the prevention and treatment of complement associated diseases, AMD and other complementary associated pathological conditions of the eye.

SUMMARY OF THE INVENTION

[0010] The invention includes methods and compositions comprising an antibody against factor Bb. In one embodiment, an anti-factor Bb antibody binds to complement factor Bb with greater affinity than complement factor B. In another aspect, an anti-factor Bb antibody binds to factor Bb and inhibits complement-dependent hemolysis. In another aspect, an anti-Bb factor antibody binds to a complement factor Bb with a Kd of less than about 1 nM. In another aspect, an anti-factor Bb antibody of the invention blocks the formation of a membrane-attacking complex (MAC).

[0011] In another embodiment, the anti-factor Bb antibody of the invention comprises a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence is (i) CDR1 selected from: (a) amino acid sequence CDR1 GDIFSSHW, SEQ ID NO: one; (b) the amino acid sequence of CDR1, which differs by no more than 2 amino acid additions, deletions or substitutions from GDIFSSHW, SEQ ID NO: 1; and (c) the amino acid sequence of CDR1 GDIFSSX ₁ W, where X ₁ is histidine and the other amino acid is replaced by alanine; (ii) CDR2 selected from: (a) the amino acid sequence of CDR2 EILPRSGITHYNENFNG, SEQ ID NO: 2; (b) the amino acid sequence of CDR2, which differs by no more than 2 amino acid additions, deletions or substitutions from EILPRSGITHYNENFNG, SEQ ID NO: 2; and (c) the amino acid sequence of CDR2 X ₁ IX ₂ PX ₃ SGITHYNENFNG, where X ₁ is glutamic acid, X ₂ is leucine, and X ₃ is arginine, and one other amino acid is replaced by alanine; and (iii) CDR3 selected from: (a) the amino acid sequence of CDR3 AINWEDS, SEQ ID NO: 3; (b) the amino acid sequence of CDR3, which differs by no more than 2 amino acid additions, deletions or substitutions from AINWEDS, SEQ ID NO: 3; and (c) the amino acid sequence of CDR3 AX ₁ NX ₂ X ₃ X ₄ S, where X ₁ is isoleucine, X ₂ is tryptophan, X ₃ is glutamic acid, X ₃ is aspartic acid, and one other amino acid is replaced with alanine; and the second amino acid sequence is (i) CDR1 selected from: (a) the amino acid sequence of CDR1 HASQNVNVWL, SEQ ID NO: 4; (b) the amino acid sequence of CDR1, which differs by no more than 2 amino acid additions, deletions or substitutions from HASQNVNVWL, SEQ ID NO: 4, SEQ ID NO: 4; and (c) the amino acid sequence of CDR1 HASQNVNVX ₁ L, where X ₁ is tryptophan and the other amino acid is replaced by alanine; (ii) CDR2 selected from: (a) the amino acid sequence of CDR2 KASNLHT, SEQ ID NO: 5; (b) the amino acid sequence of CDR2, which differs by no more than 2 amino acid additions, deletions or substitutions from KASNLHT, SEQ ID NO: 5; and (c) the amino acid sequence of CDR2 KASNLHX ₁ , where X ₁ is threonine, one other amino acid is replaced by alanine; and (iii) CDR3 selected from: (a) the amino acid sequence of CDR3 QQGQSYPYT, SEQ ID NO: 6; (b) the amino acid sequence of CDR3, which differs by no more than 2 amino acid additions, deletions or substitutions from QQGQSYPYT, SEQ ID NO: 6; and (c) the amino acid sequence of CDR3 QX ₁ GQSYPX ₂ T, where X ₁ is glutamic acid, X ₂ is tyrosine, and one other amino acid is replaced by alanine.

[0012] In another embodiment, the anti-factor Bb antibody of the invention has an amino acid sequence of a light chain variable domain that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 8-11, and an amino acid sequence of a variable a heavy chain domain that is at least 80% identical to the sequence selected from the group consisting of SEQ ID NO: 12-15. In another embodiment, the anti-factor Bb antibody of the invention has an amino acid sequence of a light chain variable domain that is at least 80% identical to a sequence selected from the group consisting of SEQ ID NO: 24-27, and an amino acid sequence of a heavy chain variable domain which is at least 80% identical to the sequence selected from the group consisting of SEQ ID NO: 28-31. In another aspect, an anti-factor Bb antibody of the invention has an amino acid sequence of a light chain variable domain selected from the group consisting of SEQ ID NO: 8-11, and an amino acid sequence of a heavy chain variable domain selected from a group consisting of SEQ ID NO: 12 -fifteen. In another aspect, an anti-factor Bb antibody of the invention has an amino acid sequence of a light chain variable domain selected from the group consisting of SEQ ID NO: 24-27 and an amino acid sequence of a heavy chain variable domain selected from a group consisting of SEQ ID NO: 28 -31. In another aspect, the anti-factor Bb antibody of the invention has the amino acid sequence of the variable domain of the light chain of SEQ ID NO: 11 and the amino acid sequence of the variable domain of the heavy chain of SEQ ID NO: 15.

[0013] In another embodiment, the anti-factor Bb antibody of the invention comprises a heavy chain and light chain variable domain selected from amino acid sequences of the light and heavy chain variable domain: SEQ ID NO: 8 / SEQ ID NO: 12; SEQ ID NO: 8 / SEQ ID NO: 13; SEQ ID NO: 8 / SEQ ID NO: 14; SEQ ID NO: 8 / SEQ ID NO: 15; SEQ ID NO: 9 / SEQ ID NO: 12; SEQ ID NO: 9 / SEQ ID NO: 13; SEQ ID NO: 9 / SEQ ID NO: 14; SEQ ID NO: 9 / SEQ ID NO: 15; SEQ ID NO: 10 / SEQ ID NO: 12; SEQ ID NO: 10 / SEQ ID NO: 13; SEQ ID NO: 10 / SEQ ID NO: 14; and SEQ ID NO: 10 / SEQ ID NO: 15; SEQ ID NO: 11 / SEQ ID NO: 12; SEQ ID NO: 11 / SEQ ID NO: 13; SEQ ID NO: 11 / SEQ ID NO: 14; and SEQ ID NO: 11 / SEQ ID NO: 15.

[0014] In another embodiment, the anti-factor Bb antibody of the invention comprises a heavy chain and light chain variable domain selected from amino acid sequences of the light and heavy chain variable domain: SEQ ID NO: 24 / SEQ ID NO: 28; SEQ ID NO: 24 / SEQ ID NO: 29; SEQ ID NO: 24 / SEQ ID NO: 30; SEQ ID NO: 24 / SEQ ID NO: 31; SEQ ID NO: 25 / SEQ ID NO: 28; SEQ ID NO: 25 / SEQ ID NO: 29; SEQ ID NO: 25 / SEQ ID NO: 30; SEQ ID NO: 25 / SEQ ID NO: 31; SEQ ID NO: 26 / SEQ ID NO: 28; SEQ ID NO: 26 / SEQ ID NO: 29; SEQ ID NO: 26 / SEQ ID NO: 30; and SEQ ID NO: 26 / SEQ ID NO: 31; SEQ ID NO: 27 / SEQ ID NO: 28; SEQ ID NO: 27 / SEQ ID NO: 29; SEQ ID NO: 27 / SEQ ID NO: 30; and SEQ ID NO: 27 / SEQ ID NO: 31.

[0015] In another aspect, the anti-factor Bb antibody of the invention is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In another aspect, an anti-factor Bb antibody of the invention is a Fab fragment, Fab 'fragment, F (ab') ₂ fragment, Fv fragment, diabody or single chain antibody molecule. In another aspect, the anti-factor Bb antibody of the invention is of type IgG1, IgG2, IgG3 or IgG4. In another aspect, an anti-factor Bb antibody of the invention is coupled to a labeling group. Such a labeling group may be an optical label, a radioisotope, a radionuclide, an enzymatic group or a biotinyl group.

[0016] In another embodiment, the invention relates to a process for producing an isolated antibody according to the invention, which comprises obtaining an antibody according to the invention from a host cell that secretes the antibody. In one aspect, this means isolating or purifying the antibody from the cell culture medium in which the host cell is grown.

[0017] In another embodiment, the invention relates to a nucleic acid molecule encoding an isolated antibody of the invention. In one aspect, a nucleic acid molecule encoding an isolated antibody of the invention is operably linked to a regulatory sequence.

[0018] In another embodiment, the invention relates to a pharmaceutical composition that comprises at least one antibody of the invention and a pharmaceutically acceptable carrier. In one aspect, the pharmaceutical composition may also contain an additional active agent.

[0019] In another embodiment, the invention relates to a method for treating or preventing a pathological condition in a patient in need of treatment or prevention, comprising administering to said patient an effective amount of at least one anti-factor Bb antibody of the invention, thereby treating or preventing a pathological condition . In one aspect, the pathological condition is an eye disease. In another aspect, the pathological condition is age-related macular degeneration (AMD).

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0020] Figure 1 illustrates a binding assay of a monoclonal antibody against factor Bb using

[0021] Figure 2 illustrates the results of a hemolysis assay using antibodies against factor B or against factor Bb in the presence of 10% normal human serum.

[0022] Figure 3 is a schematic illustration of the structure of complement factor B.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The section headings used herein are for structural purposes only and should not be construed as limiting the subject matter of this invention.

[0024] To obtain recombinant DNA, the synthesis of oligonucleotides, tissue culture and transformation, protein purification, etc. standard technology can be applied. Enzymatic reactions and purification methods can be carried out in accordance with the specifications of the manufacturer or as is usually done in the art or as described herein. The following procedures and methods can generally be carried out in accordance with traditional methods well known in the art, and as described in various general and more specialized references that are cited and discussed in the text of the description. See, e.g., Sambrook et al, 2001, Molecular Cloning:. A Laboratory Manual , 3 ^{rd ed, Cold Spring Harbor Laboratory Press} , Cold Spring Harbor, NY, which is incorporated herein by reference for any purpose.. Unless specific definitions are given, the nomenclature used in connection with and the laboratory procedures and methods of molecular biology, biological chemistry, physical and biophysical chemistry, analytical chemistry, organic chemistry and medical and pharmaceutical chemistry described in this document are those that are good known and generally applicable in the art. Standard methods can be used for chemical synthesis, chemical analysis, pharmaceutical preparation, drug production, and patient delivery and treatment.

[0025] The following definitions are used in this document:

[0026] As used herein, the term "protein" refers to at least two covalently linked amino acids and is used interchangeably with polypeptides, oligopeptides and peptides. Two or more covalently linked amino acids are linked by a peptide bond.

[0027] “Factor B” refers to a human factor B, the amino acid sequence of which is shown in SEQ ID NO: 16. Factor B, protein B, complement factor B, complement protein B belong to the same sequence as SEQ ID NO: 16 Other terms may be used to indicate factor B or variants of factor B (for example, “preproprotein B”). Factor Ba (SEQ ID NO: 17) is a single polypeptide fragment of factor B.

[0028] "Factor Bb" refers to a polypeptide fragment (SEQ ID NO: 7) of a human factor.

[0029] The terms “antibody” and “immunoglobulin” are used interchangeably in the broadest sense to mean a protein containing one or more polypeptide chains that interact with a particular antigen by binding several CDRs and an antigen epitope. An antibody may be monoclonal (e.g., full-sized or intact monoclonal antibodies), polyclonal, multivalent and / or multispecific (e.g., bispecific antibodies as long as they exhibit the desired biological activity). Antibodies can also be or include fragments of antibodies (as described herein).

[0030] The term "epitope" is used to mean a sequence, structure, or molecule that is recognized and bound by an antibody. The epitope may be called the "antigenic site."

[0031] “Antibody fragments” contain only a portion of an intact antibody, wherein this portion preferably retains at least one, preferably most or all of the functions normally associated with this portion, if present in the intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') _2, and Fv fragments; diabodies; linear antibodies; single chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment contains an antigen-binding portion of an intact antibody and, thus, retains the ability to bind antigen. In another embodiment, an antibody fragment, for example, containing an Fc region, retains at least one of the biological functions typically associated with the Fc region, if present in an intact antibody, such as FcR binding, modulation of the antibody half-life, ADCC function and complement binding. In one embodiment, an antibody fragment is a monovalent antibody that has an in vivo half-life substantially similar to an intact antibody. For example, such an antibody fragment may contain an antigen-binding arm linked to an Fc sequence capable of conferring stability to an in vivo fragment.

[0032] As used herein, a “monoclonal" antibody refers to an antibody obtained from a population of cells, wherein a population of cells is obtained by cloning from a single parent cell. Monoclonal antibodies are homogeneous antibodies, i.e. the individual antibodies that make up the population are identical in the sense that they are derived from the same genes and have the same amino acid sequence and protein structure, with the exception of possible natural mutations that may be present in small amounts, and post-translational modifications that may, in some cases, vary. Monoclonal antibodies may, in some embodiments of the invention, be highly specific. In some embodiments of the invention, the monoclonal antibody may be directed against a single antigenic site. In addition, unlike other antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against one determinant on the antigen. Individual monoclonal antibodies can be obtained by any specific method. For example, monoclonal antibodies for use in accordance with the present invention can be obtained by the hybridoma method, first described by Kohler et al. (1975) Nature 256: 495, or can be obtained by recombinant DNA methods (see, for example, US patent No. 4816567) or from phage libraries of antibodies using the methods described in Clackson et al. (1991) Nature 352: 624-628 and Marks et al. (1991) J. Mol. Biol. 222: 581-597.

[0033] The term "polyclonal" antibody is used to describe a heterogeneous population of antibodies derived from a heterogeneous population of parent antibody-producing cells. In most cases, polyclonal antibodies have different affinities for different epitopes and are derived from genes with different sequences.

[0034] "Chimeric" antibodies are antibodies containing amino acid sequences obtained from two or more different species.

[0035] Humanized antibodies are chimeric antibodies derived from a non-human parent antibody. In many cases, the specific amino acid positions in the humanized antibody have been modified to match the amino acid identity at the corresponding position in the human antibody. In many cases, positions in the variable region of the parent (non-human) antibody are replaced by amino acids from the variable region of the human species. This leads to the creation of a humanized mouse, rat, rabbit or monkey antibody with the desired specificity, affinity and properties.

[0036] "Variant" refers to sequences that contain at least one difference compared to the parent sequence. A variant polypeptide is a protein having at least about 75% amino acid sequence identity with the parent sequence. A variant protein may have at least about 80% amino acid sequence identity, or at least about 85% amino acid sequence identity, or at least about 90% amino acid sequence identity, or at least about 95% amino acid sequence identity, or at least about 98% amino acid sequence identity, or at least about 99% amino acid sequence identity with the native amino acid sequence or wild-type minacid sequence. In some cases, variant antibodies are antibodies having one or more difference (s) in amino acid sequence compared to the parent antibody. Humanized and chimeric antibodies are variant antibodies. Therefore, variant antibodies have less than 100% sequence identity with the parent antibody.

[0037] “Isolated” or “purified” refers to a molecule that has been separated and / or disposed of from at least one component of its natural environment, said component being a material that may interfere with the use or activity of the molecule. Components include peptides, sugars, nucleic acids, enzymes, hormones, and other protein or non-protein dissolved components.

[0038] “Complementarity determining regions” (CDRs) refer to one or more regions in an antibody in which residues of one or more CDRs promote antigen binding. In many cases, individual CDR amino acids may be in close proximity to the atoms of the target antigen. In some embodiments, the CDRs may be in an immunoglobulin, which may consist of three regions of CDRs. In some cases, for example, if there is more than one CDR sequence in a larger amino acid sequence, the CDRs can be separated by other sequences, and the CDRs themselves are numbered. In some cases, multiple CDRs are defined as CDR1, CDR2, and CDR3. Each CDR may contain amino acid residues from the complementarity determining region as defined by Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The numbering of amino acids of the CDR, as well as other sequences of the antibody or antibody fragment, corresponds to the numbering of Kabat. In many cases, CDRs can be determined by their position in the variable region sequence (Kabat numbering), for example, light chain CDR1 may contain an amino acid sequence between position 24 and position 33; between position 50 and position 56 in the case of LC CDR2; and between 89 and 97 in the case of LC CDR 3; a CDR of the heavy chain can be between position 26 and position 33 in the case of CDR1; 50 and 66 for HC CDR 2; and between position 97 and position 103 in the case of HC CDR 3, and / or hypervariable loops may be between light chain residues 26-32 (LC CDR1), residues 50-52 (LC CDR2) and residues 91-96 (LC CDR3); and heavy chain residues 26-32 (HC CDR1), residues 53-55 (HC CDR2) and residues 97-101 (HC CDR3). In some cases, the complementarity determining region may include amino acid residues from both the Kabat-determined CDR region and the hypervariable loop. In some embodiments of the invention, for example, in which the antibody is a single chain immunoglobulin, more than one CDR, more than two CDRs, more than three CDRs, more than four CDRs, or more than five CDRs can be present. In some embodiments of the invention, the antibody may consist of six CDRs.

[0039] “Frame regions", FR, are variable domain residues other than CDR residues. In most embodiments of the invention, the variable domain comprises from two to four FRs determined sequentially. For example, a variable region containing three CDRs contains four FRs: FR1, FR2, FR3, and FR4. If the CDRs are determined according to Kabat, the location of the FR residues of the light chain corresponds to approximately residues 1-23 (LCFR1), 34-49 (LCFR2), 57-88 (LCFR3) and 98-107 (LCFR4), and the location of the FR residues of the heavy chain corresponds to approximately residues 1-25 (HCFR1), 34-49 (HCFR2), 67-96 (HCFR3) and 104-113 (HCFR4) from the heavy chain residues. If the CDRs contain amino acid residues from hypervariable loops, the arrangement of the light chain FR residues corresponds to approximately residues 1 -23 (LCFR1), 34-49 (LCFR2), 57-88 (LCFR3) and 98-107 (LCFR4) from the light chain, and the arrangement residues of the heavy chain FR corresponds to approximately residues 1-25 (HCFR1), 34-49 (HCFR2), 67-96 (HCFR3) and 104-113 (HCFR4) of the heavy chain residues. In some cases, when the CDR contains amino acid residues from both the CDR as defined by Kabat and the residues of the hypervariable loop, the FR residues will be adjusted accordingly. For example, if HC CDR1 contains amino acids H26-H35, the heavy chain residues FR1 correspond to positions 1-25, and the FR2 residues correspond to positions 36-49.

[0040] The "variable domain" corresponds to the positions of the light chain and heavy chain of a traditional antibody molecule that contains the amino acid sequences of complementarity determining regions (CDRs) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0041] “Fv” or “Fv fragment” refers to an antibody fragment that contains a complete antigen recognition and binding site comprising FR and CDR sequences. In many embodiments, the Fv consists of a dimer of the variable domain of one heavy and one light chain, which are in close bond, which may be covalent in nature, for example, in a single chain Fv molecule (scFv). The interaction of the three CDRs of each variable domain defines an antigen binding site on the surface of the VH-VL polypeptide. Together, six CDRs or a subgroup thereof are conferred antibody antigen binding specificity. Moreover, even one variable domain (or half of the Fv containing only three CDRs specific for the antigen) has the ability in some cases to recognize and bind antigen, although usually with a lower affinity than the whole binding site.

[0042] A “Fab” or “Fab fragment” comprises a variable and constant domain (CL) of a light chain and a variable domain and a first constant domain (CH1) of a heavy chain. Antibody fragments F (ab ') ₂ contain a pair of Fab fragments that are generally covalently linked near their carboxy terminus via articulated cysteines between them. Other chemical conjugation of antibody fragments are also known in the art.

[0043] “Percentage (%) of amino acid sequence identity” is defined as the percentage of amino acid residues in a candidate sequence that are identical with amino acid residues in the control sequence after aligning the sequences and making gaps, if necessary, to achieve the maximum percentage of sequence identity, and without taking into account any conservative substitutions as part of the sequence identity. Alignment in order to determine the percent identity of amino acid sequences can be carried out in various ways that are within the competence of the art, for example, using publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. The sequence identity is then calculated with respect to the longer sequence, i.e., even if the shorter sequence shows 100% sequence identity with part of a longer sequence, the overall sequence identity will be less than 100%.

[0044] “Percentage (%) of amino acid sequence homology” is defined as the percentage of amino acid residues in a candidate sequence that are homologous to amino acid residues in the control sequence after aligning the sequences and making gaps, if necessary, to achieve the maximum percentage of sequence homology. This method takes into account conservative substitutions. Conservative substitutions are those in which substitution of an amino acid with a similar amino acid is possible. Amino acids can have similarities in several characteristics, for example, size, shape, hydrophobicity, hydrophilicity, charge, isoelectric point, polarity, aromaticity, etc. Alignment in order to determine the percentage of homology of amino acid sequences can be carried out in various ways within the competence of specialists in this field of technology. In some cases, amino acid sequences can be aligned using publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. The sequence homology with respect to the longer sequence is then calculated, i.e., even if the shorter sequence shows 100% sequence identity with part of a longer sequence, the total sequence identity will be less than 100%.

[0045] “Percentage (%) of nucleotide sequence identity” is defined as the percentage of nucleotides in a candidate sequence that are identical to the nucleotides in the control sequence after aligning the sequences and making gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment to determine the percent identity of nucleotide sequences can be carried out in various ways that are within the competence of the art, for example, using publicly available software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for alignment, including any algorithms necessary to achieve maximum alignment over the full length of the sequences being compared. The sequence identity is then calculated with respect to the longer sequence, i.e., even if the shorter sequence shows 100% sequence identity with part of a longer sequence, the overall sequence identity will be less than 100%.

[0046] The "activity" or "biological activity" of a molecule may depend on the type of molecule and the availability of methods for analyzing this activity. For example, in the context of an antibody to factor Bb, activity refers to its ability to partially or completely inhibit the biological activity of factor Bb, for example, binding to other complement proteins, serine protease activity, or MAK formation. A preferred biological activity of the inventive anti-Bb factor antibody is the ability to achieve a measurable improvement in a condition, for example, a pathology associated with a disease factor or a pathological condition, such as, for example, a complement associated pathological condition of the eyes. In some cases, the activity inhibited by the disclosed anti-Bb factor antibody is a protease or cleavage activity. In other cases, activity is the ability to bind other complement proteins to the complex. In some embodiments, the activity of the disclosed anti-Bb factor antibody is determined by its ability to inhibit hemolysis. Activity can be determined using in vitro or in vivo methods, including binding assays, the use of a relevant animal model, or human clinical studies.

[0047] The expression "complement associated pathological condition of the eyes" is used in the broadest sense and includes all pathological conditions of the eyes, the pathology of which involves complement, activated by the classical, lectin, alternative or external paths. Complementary pathological conditions of the eye include, but are not limited to, macular degenerative diseases, such as all stages of age-related macular degeneration (AMD), including dry and exudative (nonexudative and exudative) forms, choroidal neovascularization (CNV), uveitis, diabetic and other associated ischemia including diabetic macular edema, central retinal vein occlusion (OCVS), branched retinal vein occlusion (DEM) and other intraocular neovascular diseases, e as diabetic macular edema, pathological myopia, disease von Hippel-Lindau, histoplasmosis eyes, corneal neovascularization, and retinal neovascularization. A preferred group of complementary associated pathological conditions of the eye includes age-related macular degeneration (AMD), including dry and wet (non-exudative and exudative) AMD, choroidal neovascularization (CNV), macular telangiectasia, uveitis, diabetic and other associated ischemic macular degenerative neovascular disease and , pathological myopia, von Hippel-Lindau disease, eye histoplasmosis, colloidal dystrophy of the retina of Doina / Malattia Leventinese, Stargardt disease , Glaucoma, retinal vein occlusion central (CRVO), ROVS, corneal neovascularization, retinal neovascularization.

[0048] “Pharmaceutically acceptable” refers to a drug approved or approved by a federal or state regulatory authority or listed in the United States Pharmacopeia or other generally recognized pharmacopeia for use in animals, and in particular in humans.

[0049] “Pharmaceutically acceptable salt” refers to a salt of a compound that possesses the necessary pharmacological activity of the parent compound. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids, such as acetic acid, propionic acid, caproic acid, cyclopentane propionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid slots, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] -oct-2-en-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid , trimethylacetic acid, tertiary caproic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxy naphtha acid, salicylic acid, stearic acid, muconic acid and the like; and salts formed when the acidic proton present in the parent compound is replaced by a metal ion, for example, an alkali metal ion, an alkaline earth metal ion or an aluminum ion; or coordination complexes with an organic base such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine and the like. In certain embodiments of the invention, the pharmaceutically acceptable salt is a hydrochloride salt. In certain embodiments of the invention, the pharmaceutically acceptable salt is a sodium salt.

[0050] “Pharmaceutically acceptable excipient” refers to a pharmaceutically acceptable diluent, a pharmaceutically acceptable adjuvant, a pharmaceutically acceptable solution, a pharmaceutically acceptable carrier, or a combination of any of the above components with which the compound of the present invention can be administered to a patient who does not interfere with it pharmacological activity and which is non-toxic when administered in doses sufficient to provide a therapeutically effective the quality of the compound or its pharmacologically active metabolite.

[0051] “Treatment” refers to the administration of at least one therapeutic agent to prevent the development or alteration of the pathology of the disorder, or to alleviate or reduce the symptom of the disorder. Accordingly, treatment refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those who already have a disorder, as well as those who need to prevent the disorder. As disclosed herein, a preferred administration agent comprises at least one of the disclosed antibodies against factor Bb. In the treatment of a complement-related disease, a therapeutic agent containing at least one of the antibodies disclosed in the present invention or a coding sequence for such an antibody can directly or indirectly alter the response rate of the complement path component or make the disease more susceptible to treatment with other therapeutic agents, for example , antibiotics, antifungal agents, anti-inflammatory agents, chemotherapeutic agents, etc.

[0052] A “therapeutically effective amount” refers to an amount of an agent that, when administered to a subject to treat a disease or at least one of the clinical symptoms of the disease, is sufficient to effect such treatment of the disease or its symptom. A particular therapeutically effective amount may vary, for example, depending on the agent, the disease and / or symptoms of the disease, the severity of the disease and / or symptoms of the disease, the age, weight and / or health of the patient being treated, and the instructions of the attending physician. A suitable amount of any given component can be determined by those skilled in the art and / or can be determined using routine experimentation.

[0053] “Therapeutically effective dose” refers to a dose that provides an effective treatment for a disease in a patient. A therapeutically effective dose may vary from agent to agent and / or patient to patient, and may depend on factors such as the condition of the patient and the severity of the disease. A therapeutically effective dose can be determined in accordance with routine pharmacological procedures known to those skilled in the art.

[0054] The “pathology” of a disease, such as a complement associated eye disease, includes all phenomena that interfere with the well-being of the patient. These include, but are not limited to, abnormal or uncontrolled cell growth, protein production, abnormal or uncontrolled cell death, autoantibody production, complement production, complement activation, MAK formation, interfering with normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or intensification of any inflammatory or immunological responses, infiltration of inflammatory cells into the intercellular space, etc.

[0055] In the context of this document, "mammal" refers to any animal classified as a mammal, including, without limitation, humans, higher primates, domestic and farm animals, as well as animals from zoos, sports or domestic animals such as horses, pigs cattle, dogs, cats and ferrets, etc. In a preferred embodiment, the mammal is a human.

[0056] Administration “in combination with” one or more additional therapeutic agents includes simultaneous (parallel) administration and sequential administration in any order.

[0057] The present invention provides antibodies that bind a factor Bb protein.

[0058] The antibodies described herein comprise a skeleton structure with one or more complementarity determining regions (CDRs). In certain embodiments, the CDRs contain no more than two amino acid additions, deletions, or substitutions as compared to one or more of the heavy chain CDR1 and CDR2 and CDR3 of the heavy chain and CDR1 of the light chain CDR2 and CDR3 of the parent sequence. In other embodiments, CDRs are determined by consensus sequence containing common conserved amino acid sequences and variable amino acid sequences, as described herein.

[0059] In certain embodiments of the invention, the skeleton structure of antibodies to factor Bb according to the invention may be based on antibodies, including, but not limited to, monoclonal antibodies, bispecific antibodies, minutrients, domain antibodies, synthetic antibodies (eg, antibody mimetics), chimeric antibodies, humanized antibodies, fusion products of antibodies (e.g., antibody conjugates) and fragments of each of these antibodies, respectively. Various structures are further described and defined below. Antibodies to factor Bb are useful in treating the effects, symptoms and / or pathologies associated with the activity of factor Bb. These include, but are not limited to, atherosclerosis, ischemia-reperfusion after acute myocardial infarction, nephritis with Shenlein-Genoch purpura, hemorrhagic vasculitis, rheumatoid arthritis, arteritis, aneurysm, stroke, cardiomyopathy, hemorrhagic shock, damage with tissue crush hypovolemic shock and intestinal ischemia, transplant rejection, cardiac surgery, ChKTA, spontaneous miscarriage, neuronal damage, spinal cord injury, myasthenia gravis, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Guillain-Barré syndrome, Parkinson's disease, Alzheimer's disease, acute respiratory failure syndrome, asthma, chronic obstructive pulmonary disease, acute post-transfusion lung injury, acute lung damage, Goodpast disease, myocardial infarction, inflammation after the use of cardiopulmonary bypass , cardiopulmonary bypass, septic shock, graft rejection, xenograft, burn injuries, systemic lupus erythematosus, non-membranous frit, Berger's disease, psoriasis, pemphigoid, dermatomyositis, antiphospholipid syndrome, inflammatory bowel disease, hemodialysis, leukopheresis, plasmapheresis, heparin-induced extracorporeal precipitation of LDL with membrane oxygenation, extracorporeal leukopheresis membranes, hepatopheresis membranes, oxygenation, extracorporeal membrane oxygenation, and the like.

[0060] Other uses of the disclosed antibodies include, for example, the diagnosis of complement and factor Bb-associated diseases.

[0061] In aspects of the present invention, antibodies are provided against Bb factor, in particular antibodies that contain at least one CDR, including heavy and / or light chain CDRs, as more fully described below, or combinations thereof. [0062] In one aspect, antibodies to factor Bb inhibit the activity of factor Bb or inhibit the ability of factor Bb to form protein complexes. Not limited to a specific mechanism or theory, it can be said that in some embodiments of the invention, antibodies disrupt the complement pathway, thereby disrupting the complement cascade, MAC production, and cell lysis. This disorder may include, but is not limited to, dry and wet (non-exudative and exudative) AMD, choroidal neovascularization (CNV), uveitis, diabetic and other retinopathy associated with ischemia, diabetic macular edema, pathological myopia, von Hippelistoplasm Lindau disease, eyes, occlusion of the central retinal vein (OCVS), corneal neovascularization, retinal neovascularization and the like.

[0063] Thus, the antibodies of the invention can serve to detect pathological conditions associated with the complement system, or diseases or pathological conditions associated with factor Bb. In addition, antibodies can be used to regulate and / or suppress effects mediated by factor B and / or other downstream complement proteins, such as those that are effective in the treatment and prevention of various diseases or pathological conditions associated with complement and / or factor Bb. This disorder may include, but is not limited to, atherosclerosis, ischemia-reperfusion after acute myocardial infarction, nephritis with Shenlein-Genoch purpura, hemorrhagic vasculitis, rheumatoid arthritis, arteritis, aneurysm, stroke, cardiomyopathy, hemorrhagic shock, tissue damage failure, hypovolemic shock and intestinal ischemia, transplant rejection, cardiac surgery, CTDI, spontaneous miscarriage, neuronal damage, spinal cord injury, myasthenia gravis, Hunting disease she, amyotrophic lateral sclerosis, multiple sclerosis, Guillain-Barré syndrome, Parkinson's disease, Alzheimer's disease, acute respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, acute post-transfusion lung injury, acute lung damage, Goodpast disease, myocardial infarction, inflammation after application cardiopulmonary bypass, cardiopulmonary bypass, septic shock, transplant rejection, xenograft, burn injuries, systemic lupus erythematosus, membranous nephritis, Berger’s disease, psoriasis, pemphigoid, dermatomyositis, antiphospholipid syndrome, inflammatory bowel disease, hemodialysis, leukopheresis, plasmapheresis, heparin-induced extracorporeal precipitation of LDL with membrane oxygenation, extracorporeal membranous leukranapheresis membrane oxygenation, extracorporeal membrane oxygenation, and the like.

[0064] In particular, the invention provides antibodies against factor Bb and polynucleotides that encode them. In various aspects, antibodies against factor Bb inhibit at least one of the biological responses mediated by factor Bb and / or other complement proteins, and thus can be used to reduce the manifestation of complement-associated and factor Bb-associated diseases or disorders. The invention also provides expression systems, including mammalian cell lines and bacterial cells, for the production of antibodies to factor Bb and methods for treating diseases associated with factor Bb.

[0065] Antibodies of the invention comprise a skeleton structure with one or more complementarity determining regions (CDRs) that bind to factor Bb. In one embodiment, the amino acid sequence comprises any of SEQ ID NO: 1-6 or SEQ ID NO: 18-23.

[0066] In various embodiments, the antibody comprises a first and / or second amino acid sequence. In one embodiment, the first and / or second amino acid sequence is selected from the group consisting of SEQ ID NO: 8-15 or SEQ ID NO: 24-31.

[0067] In various embodiments, the antibodies may contain one or both of the first and second amino acid sequences. The first and second amino acid sequences may be a single linear amino acid sequence, may be covalently linked by disulfide bridges, or may be non-covalently linked.

Factor bb

[0068] The complement factor B is a glycosylated protein consisting of one 93,000 Da polypeptide chain encoded by the CFB gene. It is an important component of the alternative complement activation pathway and is found in human plasma at a concentration of approximately 200 μg / ml. In the presence of Mg ^++, factor B binds to C3b, and the C3b: B complex can be activated by factor D, a serine protease, which circulates as an active trypsin-like serine protease. Cleavage of factor B by factor D results in the release of the Ba fragment (33,000 Da) and leaves the Bb fragment (60,000 Da) bound to C3b. This Bb subunit is a serine protease called C3 and C5 convertase, as it converts both of these proteins into an active form by cleaving small peptides C3a and C5a, respectively.

[0069] Factor B is a highly regulated, highly specific serine protease. In activated form, it catalyzes a key complement activation step to initiate inflammatory responses, cell lysis, phagocytosis and stimulation of B cells. Factor B is activated by assembly with surface-bound C3b or its liquid phase analogue C3 (H ₂ O). After binding to C3, factor B is cleaved by factor D into a small fragment, factor Ba (residues 1-234) and a large fragment, factor Bb (residues 235-739). Fragment Ba dissociates from the complex, leaving the C3b-Bb complex of the C3 convertase of an alternative pathway that cleaves C3 into C3a and C3b. The C3b-Bb protease complex is unstable, and after dissociation from the complex, factor Bb does not reassociate with C3b.

[0070] The proenzyme factor B consists of three N-terminal domains of the complement control protein (CCP) linked by a 45-linker linker to the VWA domain, and a C-terminal domain of the serine protease (SP) that carries the catalytic center. In the active center of factor B, sharp differences are observed from other serine proteases. Factor Bb contains the C-terminal domain of the serine protease, and the CCP domains are in factor Ba.

[0071] The amino acid sequence of human factor B is given in SEQ ID NO: 16. Other forms of factor B useful in the present invention include mutants and variations that are at least 70% or at least 90% homologous to the human native sequence factor B of SEQ ID NO: 16.

[0072] The amino acid sequence of human factor Bb is represented by SEQ ID NO: 7. Other forms of factor Bb useful in the present invention include mutants and variations that are at least 70% or at least 90% homologous to the sequence of the human native factor Bb SEQ ID NO: 7.

[0073] The amino acid sequence of human factor Ba is represented by SEQ ID NO: 17. Other forms of factor Ba useful in the present invention include mutants and variations that are at least 70% or at least 90% homologous to the sequence of the human native factor Ba SEQ ID NO: 17.

GVB0) и кроличьи эритроциты. Затем инкубируют реакцию при 37°С в течение 30 минут в шейкере. Добавляют 1,0 мл холодного GVBE. Смешивают и центрифугируют в течение 3 мин приблиз. при 1000×g или выше, чтобы осадить клетки. Переносят 100 мкл супернатанта в 96-луночный планшет и считывают на 412 ни (SoftMax Pro 4.7.1). Данные анализируют при помощи GraphPad Prism 6.[0074] The inhibitory function / activity of factor B, factor Bb, or factor Ba, as described herein, represents inhibition of factor Bb. One example of an analysis of the alternative complement pathway is hemolysis analysis: activation of the alternative pathway (AP) requires higher serum concentrations than the classical pathway. In the general case, a final concentration of 5 mM Mg ⁺⁺ in the presence of 5 mM EDTA is used for analysis, while EDTA predominantly chelates Ca ⁺⁺ . AP in most mammalian species is activated spontaneously with rabbit erythrocytes, therefore, they are a convenient target. Rabbit erythrocytes (Complement Technology, Inc.) are prepared by washing 3 times with GVB0 (CompTech product) and resuspending at 5 × 10 ⁸ / ml. Different amounts of anti-factor Bb antibody were diluted with GVB0. Mix 100 μl of the reaction on ice from a series of serial dilutions of antibodies against factor Bb, 0.1 MgEDTU (CompTech product), 1/2 NPP (normal human serum, diluted

GVB0) and rabbit red blood cells. Then the reaction is incubated at 37 ° C for 30 minutes in a shaker. Add 1.0 ml of cold GVBE. Mix and centrifuge for approximately 3 minutes. at 1000 × g or higher to precipitate cells. Transfer 100 μl of the supernatant to a 96-well plate and read on 412 ni (SoftMax Pro 4.7.1). Data is analyzed using GraphPad Prism 6.

Antibodies to factor Bb.

[0075] In one aspect, antibodies are provided that bind factor Bb with greater affinity than they bind factor B.

[0076] In certain aspects, the invention provides recombinant antibodies that bind factor Bb, i.e. antibodies to factor Bb or antibodies against factor Bb. In this context, recombinant antibodies can be obtained using recombinant methods, i.e. by expression of a recombinant nucleic acid as described below. Methods and methods for producing recombinant proteins are well known in the art.

[0077] In some embodiments, the antibodies of the invention are isolated or purified. An isolated or purified antibody can be dispensed with at least some of the material with which it is usually associated in its natural state (contaminant material). In a preferred embodiment, the contaminant is less than about 50%, more preferably less than about 20%, and more preferably less than about 10% by weight relative to the total weight of the target sample. In some embodiments, the contaminant may be a protein or peptide.

[0078] Pure protein is at least about 50% by weight of the total protein, preferably at least about 80% and most preferably at least about 90%. In many embodiments, the purified anti-Bb factor antibody is obtained from an organism other than the one into which it is delivered. In some embodiments of the invention, an anti-Bb factor antibody can be obtained at a significantly higher concentration than is commonly observed by using an inducible promoter or a high expression promoter such that increased antibody concentration levels are obtained.

[0079] In some embodiments, the isolated or purified antibody can be freed from components that may interfere with the diagnostic and / or therapeutic use of the antibody. In preferred embodiments of the invention, the antibody is purified to more than 90% by weight of the antibody as determined by the Lowry method and, most preferably, to more than 99% by weight to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using conventional methods of amino acid sequencing (for example, Edman cleavage and mass spectrometry), or until homogenous with SDS-PAGE under reducing or non-reducing conditions using environmental stitching in blue coomassie or silver. Isolated antibodies include in situ antibodies in recombinant cells if at least one component of the antibody’s natural environment is missing. However, as a rule, an isolated antibody is obtained using at least one purification step.

[0080] The disclosed antibody may specifically bind to factor Bb and be used to inhibit or modulate the biological activity of factor Bb. In certain embodiments of the invention, the disclosed antibodies are created by immunizing an animal, in other cases, antibodies can be obtained using recombinant DNA technology. In further embodiments, antibodies against factor Bb can be prepared by enzymatic or chemical cleavage of naturally occurring antibodies. In some embodiments of the invention, the antibody may constitute a tetramer. In some of these embodiments, each tetramer typically consists of two identical pairs of polypeptide chains, with each pair containing one light chain (typically having a molecular weight of about 25 kDa) and one heavy chain (typically having a molecular mass about 50-70 kDa). The amino-terminal part of each chain contains a variable region from about 100 to 110 or more amino acids and may be responsible for antigen recognition. The carboxy-terminal part of each chain can define a constant region, which is mainly responsible for effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha or epsilon and determine the antibody isotype, respectively, as IgM, IgD, IgG, IgA and IgE. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3 and IgG4.

[0081] Some antibodies of natural origin, for example, antibodies that can be found in camels and llamas, may be dimers consisting of two heavy chains and not include light chains. Muldermans et al., 2001, J. Biotechnol. 74: 277-302; Desmyter et al., 2001, J. Biol. Chem. 276: 26285-26290. Crystallographic studies of camelid antibodies revealed that the CDR3 regions of these antibodies form a surface that interacts with the antigen and, thus, is critical for antigen binding, as in more typical tetrameric antibodies. The invention includes dimeric antibodies consisting of two heavy chains, or fragments thereof, which can bind to and / or inhibit the biological activity of factor Bb.

[0082] The antibodies of the invention specifically bind to a factor Bb protein, preferably human factor Bb. An antibody can specifically bind to a target antigen if the antibody has a higher binding affinity for this target antigen than for any other antigen or protein. Thus, the antibodies described herein bind with higher affinity to factor Bb than to any other protein. Typically, binding affinity is measured by determining the equilibrium binding constant, for example, K _d (or Kd) or K _a (or Ka). In some embodiments of the invention, the disclosed antibody binds to a target antigen with a CD of from about 10 ^-7 M to about 10 ^-12 M or from about 10 ^-8 M to about 10 ^-11 M, or from about 10 ^-9 M to about 10 ^-10 M. In most cases, the Kd of an open antibody with respect to a non-target antigen may be higher than Kd of a target antigen, for example, when Kd of a target is 10 ^-10 M and Kd of a non-target is 10 ^-8 M. In some cases, the Kd for another antigen is more than 1X higher than the Kd of the target antigen, more than 2X higher than the Kd of antigen a-targets, more than 3X exceeds the Kd of the target antigen, more than 4X exceeds the Kd of the target antigen, more than 5X exceeds the Kd of the target antigen, more than 6X exceeds the Kd of the target antigen, more than 7X exceeds the Kd of the target antigen, more than 8X exceeds the Kd of the target antigen, more than 9X exceeds the Kd of the target antigen, more than 10X exceeds the Kd of the target antigen (for example, when the Kd of an antibody is X ^-09 M for the target antigen, the Kd of the antibody for another antigen may be 10X longer or form X ^-08 M) or more than 100 × (e.g., when the antibody KD coc ulation X ^-10 M against the target antigen, KD of the antibody for a different antigen may be greater than or 10X X ^-08 M). In some cases, the equilibrium binding constant can be expressed as the equilibrium association constant K _a or K a.

, Menlo Park, CA, USA) в соответствии с указаниями производителя. Другие типы анализа связывания белка включают: совместную иммунопреципитацию; бимолекулярную комплементацию флуоресценции; аффинный электрофорез; анализ с осаждением; перенос метки; дрожжевой двугибридный скрининг; фаговый дисплей; in vivo перекрестное связывание белковых комплексов с применением фотореактивных аминокислотных аналогов; тандемную аффинную очистку; химическое перекрестное связывание; химическое перекрестное связывание с последующей высокой массовой масс-спектрометрией MALDI; SPINE (эксперимент с взаимодействием со стреппротеином); количественную преципитацию в комбинации с нокдауном; метод близкого лигирования с биослойной интерферометрией; двойную интерферометрию поляризации; статическое светорассеяние; динамическое светорассеяние; поверхностный плазмонный резонанс; поляризацию/анизотропию флуоресценции; флуоресцентную корреляционную спектроскопию; резонансный перенос энергии флуоресценции; определение активности белка по ЯМР-измерениям многоядерной релаксации или 2D-ПФ ЯМР-спектроскопию в растворах в комбинации с нелинейным регрессионным анализом ЯМР-релаксации или групп данных 2D-ПФ-спектроскопии; стыковку белое-белок; изотермическую титрационную калориметрию; и микромасштабный термофорез.[0083] The equilibrium binding constant can be determined in various ways. In some cases, the equilibrium binding constant for the disclosed antibody is determined by measuring the rates of association (k ₁ ) and dissociation (k _-1 ) in the protein binding assay. One typical method for measuring the equilibrium binding constant is biolayer interferometry (BLI). BLI is a labelless technology that enables the determination of binding kinetics in solution. In one exemplary method, the antibody may be human IgG, and antibody capture may occur on the capture tips of a biosensor with anti-human IgG Fc (ANS) (

, Menlo Park, CA, USA) as directed by the manufacturer. Other types of protein binding assays include: co-immunoprecipitation; bimolecular complementation of fluorescence; affinity electrophoresis; analysis with precipitation; Label wrapping yeast two-hybrid screening; phage display; in vivo cross-linking of protein complexes using photoreactive amino acid analogues; tandem affinity purification; chemical crosslinking; chemical crosslinking followed by high mass MALDI mass spectrometry; SPINE (experiment with interaction with streptrotein); quantitative precipitation in combination with knockdown; close ligation method with biolayer interferometry; double polarization interferometry; static light scattering; dynamic light scattering; surface plasmon resonance; polarization / anisotropy of fluorescence; fluorescence correlation spectroscopy; resonant fluorescence energy transfer; determination of protein activity by NMR measurements of multinuclear relaxation or 2D PF NMR spectroscopy in solutions in combination with non-linear regression analysis of NMR relaxation or data groups of 2D PF spectroscopy; docking white-protein; isothermal titration calorimetry; and micro-scale thermophoresis.

[0084] In embodiments of the invention in which the antibody is used in therapeutic applications, one of the characteristics of an antibody to factor Bb is that it can modulate and / or inhibit one or more biological activities of factor Bb or factor Bb-mediated. In this case, the antibody can specifically bind to factor Bb, can significantly modulate the activity of factor Bb, and / or can inhibit the binding of factor Bb to other proteins (e.g., factor C3). In some cases, the antibody can inhibit the serine protease activity of factor Bb by at least about 20%, 40%, 60%, 80%, 85% or more.

GVB0) и кроличьи эритроциты. Затем инкубируют реакцию при 37°С в течение 30 минут в шейкере. Добавляют 1,0 мл холодного GVBE. Смешивают и центрифугируют в течение 3 мин приблиз. при 1000×g или выше, чтобы осадить клетки. Переносят 100 мкл супернатанта в 96-луночный планшет и считывают на 412 ни (SoftMax Pro 4.7.1). Данные анализировали при помощи GraphPad Prism 6.[0085] In many embodiments of the invention, the activity of factor Bb and the ability of an antibody to inhibit this activity is determined by analyzing lysis of red blood cells in the presence of 10% human serum. Alternative pathway activation (AP) requires higher serum concentrations than the classic pathway. In the general case, a final concentration of 5 mM Mg ⁺⁺ in the presence of 5 mM EDTA is used for analysis, while EDTA predominantly chelates Ca ⁺⁺ . AP in most mammalian species is activated spontaneously with rabbit erythrocytes, therefore, they are a convenient target. Rabbit erythrocytes (Complement Technology, Inc.) are prepared by washing 3 times with GVB0 (CompTech product) and resuspending at 5 × 10 ⁸ / ml. Different amounts of anti-factor Bb antibody were diluted with GVB0. Mix 100 μl of the reaction on ice from a series of serial dilutions of antibodies against factor Bb, 0.1 M MgEDTU (CompTech product), 1/2 NPP (normal human serum, diluted

GVB0) and rabbit red blood cells. Then the reaction is incubated at 37 ° C for 30 minutes in a shaker. Add 1.0 ml of cold GVBE. Mix and centrifuge for approximately 3 minutes. at 1000 × g or higher to precipitate cells. Transfer 100 μl of the supernatant to a 96-well plate and read on 412 ni (SoftMax Pro 4.7.1). Data was analyzed using GraphPad Prism 6.

[0086] Not every antibody that specifically binds to an antigen can block the binding of an antigen to its normal ligand and thus inhibit or modulate the biological effects of the antigen. As is known in the art, such an action may depend on which part of the antigen the antibody binds to, as well as the absolute and relative concentrations of the antigen and antibody, in this case, the anti-Bb factor antibody. To be considered capable of inhibiting or modulating the biological activity of factor Bb, as implied herein, an antibody may be able, for example, to inhibit the serine protease activity of factor Bb or human serum-mediated hemolysis by at least about 20%, 40%, 60%, 80 %, 85%, 90%, 95%, 99% or more.

[0087] The antibody concentration required to inhibit the activity of factor Bb can vary greatly and may depend on how strongly the antibody binds to factor Bb. For example, to inhibit biological activity, one antibody molecule or less per factor Bb molecule may be sufficient. In some embodiments of the invention, inhibition of the biological activity of factor Bb may require an antibody to factor Bb ratio of about 1000: 1 to about 1: 1000, including about 2: 1, 1: 1, 1: 2, 1: 4, 1 : 6, 1: 8, 1:10, 1:20, 1:40, 1:60, 1: 100, 1: 500, 1: 1000 or more. In many cases, the ability to inhibit the activity of factor Bb may depend on the concentration of factor Bb and / or the concentration of antibodies to factor Bb.

[0088] In some embodiments, the antibodies of the invention comprise (a) a framework and (b) one or more CDRs, regions that determine antigen binding specificity and affinity. Complementarity determining regions or CDRs are antibody regions that constitute the main surface contact points for antigen binding. One or more CDRs are included in the frame structure of the antibody. The skeleton structure of antibodies according to the invention may be an antibody skeleton or fragment or variant thereof, or may be completely synthetic in nature. Various framework structures of antibodies of the invention are further described herein.

[0089] In a preferred embodiment of the disclosed antibodies, the antibody may be a variant antibody having an amino acid sequence with at least 75% identity or similarity to the amino acid sequence of the parent antibody. For example, in some embodiments of the invention, the sequence of the variable domain of the light or heavy chain of an antibody variant is 75% identical to the original sequence of the variable domain of the heavy or light chain, alternatively at least 80%, alternatively at least 85%, alternatively at least 90% and alternatively at least 95%. In most cases, the variant antibody contains few or no changes in the CDR sequence and, therefore, in most cases it will bind to the target antigen with the same affinity. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in a variant sequence that are identical (i.e., the same residue) or similar (i.e., an amino acid residue belonging to the same group based on common properties side chains, see below) with the amino acid sequence of the parent antibody after aligning the sequences and introducing, if necessary, gaps to achieve the maximum percentage of identity and sequences. None of the N-terminal, C-terminal or internal extensions, deletions or insertions in the sequence of antibodies outside the variable domain should not be considered such that affect the identity or similarity of the sequences.

CDR

[0090] The antibodies of the invention comprise framework regions and one or more CDRs. An antibody according to the invention may contain from one to six CDRs (as naturally occurring antibodies of natural origin), for example, one heavy chain CDR1 ("HC CDR1" or "HC CDR1") and / or one heavy chain CDR2 ("HC CDR2" or “HC CDR2”), and / or one heavy chain CDR3 (“HC CDR3” or “HC CDR3”), and / or one light chain CDR1 (“LC CDR1” or “LC CDR1”), and / or one light CDR2 chains (“LC CDR2” or “LC CDR2”), and / or one light chain CDR3 (“LC CDR3” or “LC CDR3”). Used in the text of the description, the term "natural origin" in connection with biological materials, such as polypeptides, nucleic acids, host cells and the like, refers to materials that can be found in nature. In naturally occurring antibodies, a heavy chain CDR1 typically contains from about five (5) to about seven (7) amino acids, a heavy chain CDR2 typically contains from about sixteen (16) to about nineteen (19) amino acids, and a CDR3 the heavy chain typically contains from about three (3) to about twenty-five (25) amino acids. CDR1 of the light chain typically contains from about ten (10) to about seventeen (17) amino acids, CDR2 of the light chain is typically contains about seven (7) amino acids, and light chain CDR3 typically contains from about seven (7) to about ten (10) ami acids.

[0091] Amino acids of the present invention include natural and synthetic amino acids (eg, homophenylalanine, citrulline, ornithine and norleucine). Such synthetic amino acids may be included, in particular when the antibody is synthesized in vitro by conventional methods well known in the art. In addition, any combination of peptidomimetic, synthetic and natural residues / structures can be used. Amino acids include amino acid residues such as proline and hydroxyproline. The amino acid “R-group” or “side chain” can be in either the (L) - or (S) -configuration. In a specific embodiment, the amino acids are in the (L) or (S) configuration. In some embodiments, amino acids can form peptidomimetic structures, i.e. peptide or protein analogues, such as peptoids (see, Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89: 9367, incorporated herein by reference), which may be resistant to proteases or other physiological conditions and / or storage conditions.

[0092] The structure and properties of CDRs in an antibody of natural origin are described below. Briefly, in a conventional framework, CDR antibodies are included within the framework region in the variable region of the heavy and light chains, where they form regions responsible for antigen binding and recognition. The variable region contains at least three heavy or light chain CDRs (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, MD; see also Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917; Chothia et al., 1989, Nature 342: 877-883) within the framework region (framework regions are designated 1-4, FR1, FR2, FR3 and FR4 according to Kabat et al., 1991; see also Chothia and Lesk, 1987). In this case, the CDRs proposed in the present invention can be used not only to determine the antigen binding domain of a traditional antibody structure, but can also be included in a large number of other frame structures, as described herein.

[0093] Alanine scanning was used to determine amino acid positions in CDR sequences, which, when modified, change the binding affinity of antibodies against factor Bb.

[0094] Specific CDRs for use in the disclosed antibodies are presented in Table 1, amino acids are highlighted, the substitution of which with alanine significantly reduced binding.

[0095] The sequences of factor B, factor Ba and factor Bb are shown in Table 2.

[0096] In another embodiment, the invention provides an antibody that binds Factor Bb (SEQ ID NO: 7), said antibody comprising at least one HC CDR region containing at most two (2) amino acid additions, deletions or substitutions any of SEQ ID NO: 1-3 or SEQ ID NO: 18-20, and / or at least one LC CDR region containing at most two (2) amino acid additions, deletions or substitutions for any of SEQ ID NO: 4- 6 or SEQ ID NO: 21-23. Various variable regions of the heavy chain and light chain according to the invention are illustrated in TABLE 3 and SEQ ID NO: 8-15 or SEQ ID NO: 24-31. In some embodiments of the invention, specifically antibodies with a region of HC CDR3 or LC CDR3 are used. In addition, in some embodiments, the antibodies may contain one CDR containing no more than two (2) amino acid additions, deletions, or substitutions of a sequence selected from HC CDR regions of any of SEQ ID NO: 1-3 or SEQ ID NO: 18- 20, and an LC CDR containing no more than two (2) amino acid additions, deletions or substitutions of any of SEQ ID NO: 4-6 or SEQ ID NO: 21-23 (for example, an antibody contains two CDR regions, one HC CDR and one LC CDR, and a specific embodiment of the invention are antibodies with both HC CDR3 and LC CDR3, for example, SEQ ID NO: 3 and 6).

Variant CDR Sequences

[0097] In an additional aspect of the invention, there is provided an isolated antibody that binds Bb factor, wherein the isolated antibody contains a heavy chain amino acid sequence containing no more than two (2) amino acid additions, deletions or substitutions of any of SEQ ID NO: 12-15 or SEQ ID NO: 28-31, or an amino acid sequence of a light chain containing no more than two (2) amino acid additions, deletions or substitutions of any of SEQ ID NO: 8-11 or SEQ ID NO: 24-27.

[0098] In an additional aspect of the invention, there is provided an isolated antibody that binds Bb factor, wherein the isolated antibody contains a heavy chain amino acid sequence containing no more than two (2) amino acid additions, deletions or substitutions of any of SEQ ID NO: 12-15 or SEQ ID NO: 28-31, and the amino acid sequence of the light chain containing no more than two (2) amino acid additions, deletions or substitutions of any of SEQ ID NO: 8-11 or SEQ ID NO: 24-27. It should be noted that any of the heavy chain sequences can be combined and paired with any of the light chain sequences.

[0099] In another embodiment, the invention provides an antibody that binds factor Bb, wherein said antibody contains at least one region of HC CDRs containing no more than two (2) amino acid additions, deletions, or substitutions of any of the regions of HC CDR1, HC CDR2 or HC CDR3 (as described above) SEQ ID NO: 1-3 or SEQ ID NO: 18-20, and / or at least one LC CDR region containing at most two (2) amino acid additions, deletions, or any substitutions from the regions of LC CDR1, LC CDR2 or LC CDR3 (as described above) SEQ ID NO: 4-6 or SEQ ID NO: 21-23. In this embodiment, specifically antibodies with a region of HC CDR3 or LC CDR3 are used. In further embodiments, antibodies are used with one CDR containing no more than 2 amino acid additions, deletions or substitutions of a sequence selected from HC CDR regions of any of SEQ ID NO: 1-3 or SEQ ID NO: 18-20, and an LC CDR region containing no more than two (2) amino acid additions, deletions or substitutions of any of SEQ ID NO: 4-6 or SEQ ID NO: 21-23 (for example, an antibody contains two CDR regions, one HC CDR and one LC CDR, and a specific an embodiment of the invention are antibodies with both regions of HC CDR3 and LC CDR3, for example, SEQ ID NO: 3 and 6).

[00100] As those skilled in the art will understand, for any antibody with one or more CDRs from the illustrated sequences, any combination of CDRs independently selected from the illustrated sequences can be used. Thus, it is possible to create antibodies with one, two, three, four, five or six independently selected CDRs. Moreover, as is clear to those skilled in the art, in specific embodiments of the invention, CDR combinations are used that are not repeated, for example, in the general case, antibodies with two regions of HC CDR2 are not obtained, etc.

[00101] In an additional aspect of the invention, there is provided an isolated antibody that binds Bb factor, wherein the isolated antibody contains a heavy chain amino acid sequence containing no more than two (2) amino acid additions, deletions or substitutions of any of SEQ ID NO: 12-15 or SEQ ID NO: 28-31, or an amino acid sequence of a light chain containing no more than two (2) amino acid additions, deletions or substitutions of any of SEQ ID NO: 8-11 or SEQ ID NO: 24-27.

[00102] In an additional aspect of the invention, there is provided an isolated antibody that binds Bb factor, wherein the isolated antibody contains a heavy chain amino acid sequence containing at most two (2) amino acid additions, deletions or substitutions of any of SEQ ID NO: 12-15 or SEQ ID NO: 28-31, and the amino acid sequence of the light chain containing no more than two (2) amino acid additions, deletions or substitutions of any of SEQ ID NO: 8-11 or SEQ ID NO: 24-27. It should be noted that any of the heavy chain sequences can be combined and paired with any of the light chain sequences.

[00103] In general, the amino acid homology, similarity or identity between the individual variant CDRs described herein is at least 80% when compared with the sequences disclosed herein. In various cases, amino acid homology, similarity or identity is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99%.

Sequence Identity / Homology

[00104] As is known in the art, a large number of different programs can be used to determine the degree of sequence identity or similarity of a protein or nucleic acid with respect to a known sequence.

[00105] In the case of amino acid sequences, sequence identity and / or similarity is determined using standard methods known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2: 482, alignment algorithm for determining sequence identity by Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443, authorship similarity search method Pearson and Lipman, 1988, Proc. Nat. Acad. Sci. U.S.A. 85: 2444, computerized embodiments of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Drive, Madison, Wis.), The Best Fit sequence program described by Devereux et al., 1984 , Nucl. Acid Res. 12: 387-395, preferably used with default parameters or a selection method. Preferably, the percent identity is calculated using FastDB based on the following parameters: penalty for mismatch 1; gap penalty 1; penalty for gap size 0.33; and Compound Fee 30, "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

[00106] An example of an applicable algorithm is PILEUP. PILEUP creates multiple sequence alignment from a group of related sequences using progressive pairwise alignment. It is also possible to build a tree showing the group relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of authorship Feng & Doolittle, 1987, J. Mol. Evol. 35: 351-360; this method is similar to that described by Higgins and Sharp, 1989, CABIOS 5: 151-153. The applicable PILEUP parameters include the default gap weight of 3.00, the default gap length weight of 0.10, and weighted end gaps.

[00107] Another example of an applicable algorithm is the BLAST algorithm described in: Altschul et al., 1990, J. Mol Biol. 215: 403-410; Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402; and Karin et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90: 5873-5787. In particular, the BLAST program used is the WU-BLAST-2 program, which was obtained from Altschul et al., 1996, Methods in Enzymology 266: 460-480. WU-BLAST-2 uses several search parameters, most of which are set by default. The corrected parameters for proteins are set with the following values: overlap length = 1, overlap fraction = 0.125, threshold segment length, T = 11. The parameters HSP S and HSP S2 are dynamic values and are set by the program itself depending on the composition of a specific sequence and the composition of a specific database against which a sequence of interest is searched; however, these values can be adjusted to increase sensitivity.

[00108] An additional applicable algorithm is a BLAST with gaps described by Altschul et al., 1993, Nucl Acids Res. 25: 3389-3402. The BLAST with gaps uses the BLOSUM-62 replacement weight matrix; threshold parameter T is set to 9; a two-coincidence method for launching extensions without gaps, the cost of a gap length k worth 10 + k; X _{u is} set to 16, and X _{g is} set to 40 at the database search stage and 67 at the output stage of the algorithms. Gap alignment starts at an estimate of approximately 22 bits.

[00109] In general, amino acid homology, similarity or identity between individual variant CDRs or variable regions is at least 80% with respect to the sequences illustrated herein, and typically with preferably increasing homology or identity of at least 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and almost 100%.

[00110] Similarly, the percentage (%) of nucleotide sequence identity with respect to the nucleotide sequence of the disclosed antibody is the percentage of nucleotide residues in the candidate sequence that are identical to the nucleotide residues in the coding sequence of the antibody. In a particular method, a BLASTN WU-BLAST-2 module is used with default parameters, overlap length and overlap fraction set to 1 and 0.125, respectively.

[00111] In general, the nucleotide homology, similarity or identity between nucleotide sequences encoding individual variant CDRs and variant variable domain sequences is at least 80%, and typically with preferably increasing homology or identity of at least 85% , 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and almost 100%. In many cases, non-identical nucleotide sequences, due to the degeneracy of the genetic code, can encode the same amino acid sequence.

[00112] Homology between nucleotide sequences is often determined by their ability to hybridize with each other. In some embodiments, selective hybridization may relate to high specificity binding. Polynucleotides, oligonucleotides and fragments thereof in accordance with the invention selectively hybridize to nucleic acid chains under hybridization and washing conditions that minimize a significant amount of detectable binding to non-specific nucleic acids. As is known in the art and as discussed herein, high stringency conditions can be used to obtain selective hybridization conditions.

[00113] A person skilled in the art can easily determine the stringency of hybridization, which is generally calculated empirically based on probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes require lower temperatures. In the general case, hybridization depends on the ability of the denatured DNA to re-anneal when the complementary chains are in a medium below their melting point. The higher the degree of necessary homology between the probe and the hybridizable sequence, the higher the relative temperature can be used. It follows that higher relative temperatures tend to increase the severity of the reaction conditions, while lower temperatures tend to decrease. See Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995) for further details and an explanation of the stringency of hybridization reactions.

[00114] High stringency conditions are known in the art; see, for example, Sambrook et al., 2001, supra, and Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992, both of which are incorporated herein by reference. Severe conditions depend on the sequences and will vary in different circumstances. Longer sequences specifically hybridize at higher temperatures. An extended guide to nucleic acid hybridization can be found in Tijssen, Techniques In Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).

[00115] In some embodiments of the invention, stringency or high stringency conditions can be determined by the fact that: (1) low ionic strength and high temperature are used to flush, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1 % sodium dodecyl sulfate at 50 ° C; (2) during the hybridization, a denaturing agent such as formamide, for example, 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% ficoll / 0.1% pol ivinylpyrrolone don / 50 mM, is used sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42 ° C; or (3) apply 50% formamide, 5X SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5X Denhardt's solution, fragmented DNA from milk salmon (50 μg / ml), 0.1% SDS and 10% dextran sulfate at 42 ° C with washing at 42 ° C in 0.2X SSC (sodium chloride / sodium citrate) and 50% formamide at 55 ° C followed by washing with high rigidity consisting of 0.1X SSC containing EDTA at 55 ° C.

[00116] In general, stringent conditions are chosen so that the temperature is about 5-10 ° C lower than the melting temperature (Tm) for a particular sequence at a specific ionic strength and pH. Tm represents the temperature (at certain ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence in equilibrium (if the target sequences are in excess, at Tm 50% probes in equilibrium). Severe conditions are those in which the salt concentration is less than about 1.0 M sodium ions, typically from about 0.01 to 1.0 M sodium ions (or other salts) at a pH of from 7.0 to 8.3 and the temperature is at least about 30 ° C for short probes (for example, from 10 to 50 nucleotides) and at least about 60 ° C for long probes (for example, more than 50 nucleotides). Severe conditions can also be achieved by adding destabilizing agents such as formamide.

[00117] In another embodiment, less stringent conditions apply; for example, as is known in the art, conditions of moderate or low stringency can be applied; see, Sambrook et al., 2001, supra; Ausubel et al., 1992, supra, and Tijssen, 1993, supra.

[00118] In some cases, moderate stringency conditions may include the use of a washing solution and hybridization conditions (eg, temperature, ionic strength and% SDS) that are less stringent than those described above. An example of moderate stringency conditions is overnight incubation at 37 ° C in a solution containing: 20% formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt’s solution, 10% dextran sulfate and 20 mg / ml fragmented DNA from salmon milk, followed by washing the filters in 1X SSC at about 37-50 ° C. One skilled in the art knows how to adjust temperature, ionic strength, etc. if necessary, consider factors such as the length of the probes and the like.

[00119] In some embodiments of the invention, the disclosed antibodies and their variants can be obtained using site-specific mutagenesis of nucleotides in the DNA sequence encoding the antibody. This can be accomplished using a cassette or PCR mutagenesis or other methods well known in the art to obtain DNA encoding a variant, and then expressing recombinant DNA in cell culture, as described herein. In some cases, antibody fragments containing variant CDRs containing up to about 100-150 residues can be obtained by / and vitro synthesis using known methods. These variant fragments can exhibit the same qualitative biological activity as an analogue of natural origin, for example, binding to factor Bb and inhibition of complement, although it is also possible to choose variants that have modified characteristics, as described more fully below.

[00120] While the site or region for introducing amino acid sequence variation is specific, there is no need to predefine the mutation per se. For example, in order to optimize a mutation in a given region, random mutagenesis can be performed in the target codon or target region and the expressed CDR sequence or variable region of the antibody can be screened for the optimal required antibody activity. Methods for introducing substitution mutations into predetermined regions in DNA having a known sequence are well known, for example, mutagenesis with M13 primer and PCR mutagenesis. Mutants are screened using an antibody activity assay, such as factor Bb binding.

[00121] Amino acid substitutions are usually carried out in one residue; insertions typically occupy from about one (1) to about twenty (20) amino acid residues, although significantly larger insertions are acceptable. The range of deletions is from about one (1) to about twenty (20) amino acid residues, although in some cases deletions can be much larger.

[00122] To obtain the final derivative or variant, substitutions, deletions, insertions, or any combination thereof can be used. In general, these changes are carried out in several amino acids in order to minimize the change in the molecule, in particular, the immunogenicity and specificity of the antibody. However, in certain circumstances large changes are permissible. Conservative substitutions are generally carried out in accordance with the following scheme, shown in the form of Table 4.

[00123] Changes in functions or immunological features can be obtained by choosing substitutions that are less conservative than those listed in Table 4. For example, substitutions can be made that more significantly affect: the structure of the polypeptide skeleton in the site of change, for example, alpha-helical or beta fold structure; charge or hydrophobicity of the molecule in the target area; or side chain volume. Substitutions that, in the general case, should lead to the greatest changes in the properties of the polypeptide, are those in which (a) a hydrophilic residue, for example, seryl or threonyl, is replaced by a hydrophobic residue, for example, leucyl, isoleucil, phenylalanil, valil or alanyl; (b) cysteine or proline is substituted for any other residue; (c) a residue having an electropositive side chain, for example, lysyl, arginyl or histidyl, is replaced by an electronegative residue, for example, glutamyl or aspartyl; or (d) a residue having a bulky side chain, for example phenylalanine, is substituted with a residue without a side chain, for example glycine.

[00124] Variants typically exhibit the same qualitative biological activity and elicit the same immune response as an analogue of natural origin, although options may be chosen if necessary to modify the characteristics of the disclosed Bb factor antibody. Alternatively, a variant may be selected in which the biological activity of the disclosed antibody is altered. For example, glycosylation sites may be altered or removed, as discussed herein.

[00125] The polypeptide sequences homologous to SEQ ID NO: 1-6 and 8-15 and SEQ ID NO: 18-23, and SEQ ID NO: 24-31 are disclosed herein. The polypeptides disclosed herein may contain amino acid sequences identical to the disclosed amino acid sequences. In other cases, the claimed polypeptides contain amino acid sequences that may contain conservative amino acid substitutions compared to the disclosed sequence. Conservative amino acid substitutions may include amino acids that have similar characteristics to a substituted amino acid. In various cases, a conservative substitution can be made without a significant change in the structure or function of the polypeptide.

[00126] Conservative amino acid substitutions can be made based on the relative similarity of the side chain, size, charge, hydrophobicity, hydrophilicity, isoelectric point, etc. In various cases, the effect of substitutions on protein function can be assessed using routine studies. Conservative amino acid substitutions include amino acids with a similar hydrophilicity value, for example, when the amino acids have a hydrophobicity index, which can be based on hydrophobicity and charge of the amino acid. In various cases, conservative amino acid substitutions can be made between two amino acids of the same class, for example, non-polar amino acids, acid amino acids, basic amino acids and neutral amino acids. Conservative substitutions can also be based on size or volume. Amino acids can also be classified based on their ability to form or destroy a given structure, such as an alpha helix, beta folding, or inter- or intramolecular interaction. In various cases, conservative amino acid substitutions are based on more than one characteristic.

[00127] The polypeptides disclosed herein may contain amino acids of both natural and non-natural origin. In various cases, the side chains of naturally occurring amino acids can be replaced by side chains of non-natural origin. In various cases, amino acids can be derivatized.

[00128] The disclosed polypeptides include polypeptides that are homologous to the sequences of SEQ ID NO: 1-6 and 8-15, and SEQ ID NO: 18-31. Homology can be expressed as% identity or% similarity, or% positive results. In various cases,% identity represents the percentage of amino acids that are identical for the two aligned sequences, and% similarity or% of positive results represents the percentage of amino acids that are not identical but represent conservative substitutions. A conservative substitution may be the replacement of an amino acid with a similar charge, amino acids with a similar size, amino acids with a similar polarity, etc. For example, if you take into account the charge, then the replacement of lysine with arginine can be considered a conservative substitution.

[00129] In various cases, alignment of two polypeptides can be carried out using algorithms, for example, BLASTp. In various cases, BLASTp parameters can be set with the maximum length of the target sequence equivalent to greater or less than the length of the longest of the two polypeptides, the expected threshold can be set to 10, the segment length to 3, and the replacement matrix can be BLOSUM62 with a gap cost 11 for deposit and 1 for renewal. BLASTp can represent the homology of aligned polypeptides in the form of "identity" or "number of positive results." Aligned sequences may include gaps to achieve alignment.

[00130] In various cases, the homology of amino acid sequences may display a percentage of identity or positive results with optimal alignment, as described above. In various cases,% homology (% positive results) or% identity can be calculated by dividing the number of aligned amino acids in the comparison window. The comparison window may be the full length of one or the other polypeptide, if the length of the two polypeptides is not the same. In other cases, the comparison window may form part of one of the polypeptides. In various cases, the comparison window for determining the homology or identity of two polypeptide sequences is more than about 40 ak (amino acids), 45 ak, 50 ak, 55 ak, 60 ak, 65 ak, 70 ak, 75 ak, 80 ak, 85 ak. 90 ak, 95 ak, 100 ak, 150 ak or 200 ak, and / or less than about 200 ak, 150 ak, 100 ak, 95 ak, 90 ak, 85 ak, 80 ak, 75 ak, 70 ak, 65 ak 60 ak, 55 ak, 50 ak or 45 ak. In some embodiments of the invention, as is the case with CDR sequences, the comparison window may be less than 40 ak, for example, between less than about 25 ak, 24 ak, 23 ak, 22 ak, 21 ak, 20 ak, 19 ak, 18 ak, 17 ak, 16 ak, 15 ak, 14 ak, 13 ak, 12 ak, 11 ak, 10 ak, 9 ak, 8 ak, 7 ak, 6 ak, 5 ak or 4 ak, and more than about 3 ak , 4 ak, 5 ak, 6 ak, 7 ak, 8 ak, 9 ak, 10 ak, 11 ak, 12 ak, 13 ak, 14 ak, 15 ak, 16 ak, 17 ak, 18 ak, 19 ak, 20 ak, 21 ak, 22 ak, 23 ak or 24 ak.

[00131] In various cases, the claimed amino acid sequences may have% identity or% homology (% positive results) in a given comparison window, which is more than about 60%, 65%, 70%, 75%, 80%, 85%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and / or less than about 100%, 99%, 98%, 97%, 96%, 95 %, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75% or 70%.

[00132] In various cases, sequence alignment can be performed using various algorithms, including dynamic, local, and global alignment. For example, Smith and Waterman, 1981, Adv. Appl. Math 2: 482; Alignment Algorithm Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443; affinity method Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85: 2444. In various cases, these algorithms can be implemented by computer programs (such as EMBOSS, GAP, BESTFIT, FASTA, TFASTA BLAST, BLOSUM, etc.).

[00133] In alternative cases, conservative amino acid substitutions can be made in which the amino acid residue is replaced by another from the same class, for example, in which the amino acids are divided into non-polar, acid, basic and neutral classes as follows: non-polar: Ala, Val, Leu, Ile Phe, Trp, Pro, Met; acidic: Asp, Glu; main: Lys, Arg, His; neutral: Gly, Ser, Thr, Cys, Asn, Gln, Tyr.

[00134] In some cases, it is possible to carry out conservative amino acid substitutions in which the amino acid residue is replaced by another having a similar hydrophilicity value (for example, within plus or minus 2.0), when the next may be an amino acid with a hydrophobicity index of about -1.6, such as Tyr (-1.3) or Pro (-1.6). The following values are assigned to amino acids: Arg (+3; 0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (O); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); Ile (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4).

[00135] In alternative cases, conservative amino acid substitutions can be made in which the amino acid residue is replaced by another having a similar hydrophobicity index (for example, within plus or minus 2.0). In such cases, each amino acid can be assigned a hydrophobicity index based on its hydrophobicity and charge characteristics as follows: Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5).

[00136] In alternative cases, conservative amino acid substitutions include changes in consideration of hydrophilicity or hydrophobicity, size or volume or charge. In the general case, amino acids can be characterized as hydrophobic or hydrophilic, depending mainly on the properties of the amino acid side chain. Hydrophobic amino acids exhibit hydrophobicity greater than zero, and hydrophilic amino acids exhibit hydrophilicity less than zero based on a normalized consensus assessment of hydrophobicity according to Eisenberg et al. (J. Mol. Bio. 179: 125-142, 184). Genetically encoded hydrophobic amino acids include Gly, Ala, Phe, Val, Leu, Ile, Pro, Met, and Trp, and genetically encoded hydrophilic amino acids include Thr, His, Glu, Gin, Asp, Arg, Ser, and Lys. Non-coded genetically hydrophobic amino acids include t-butylalanine, while non-coded genetically hydrophilic amino acids include citrulline and homocysteine.

[00137] Hydrophobic or hydrophilic amino acids can be further divided based on the characteristics of their side chains. For example, an aromatic amino acid is a side chain amino acid containing at least one aromatic or heteroaromatic ring, which may contain one or more substituents, such as —OH, —SH, —CN, —F, —Cl, —Br, —I , -NO ₂ , -NO, -NH ₂ , -NHR, -NRR, -C (O) R, -C (O) OH, -C (O) OR, -C (O) NH ₂ , -C ( O) NHR, —C (O) NRR, etc., where R independently is (C ₁ -C ₆ ) alkyl, substituted (C ₁ -C ₆ ) alkyl, (C ₀ -C ₆ ) alkenyl, substituted (C ₁ -C ₆ ) alkenyl, (C ₁ -C ₆ ) alkynyl, substituted (C ₀ -C ₆ ) alkynyl, (C ₅ -C ₂₀ ) aryl, substituted (C ₀ -C ₂₀ ) aryl, (C ₆ -C ₂₆ ) alkaryl, substituted (C ₆ -C ₂₆ ) alkaryl, 5-2 0-membered heteroaryl, substituted 5-20-membered heteroaryl, 6-26-membered alkheteroaryl or substituted 6-26-membered alkheteroaryl. Genetically encoded aromatic amino acids include Phe, Tyr and Trp.

[00138] A non-polar or apolar amino acid is a hydrophobic amino acid with a side chain that is uncharged at a physiological pH and that contains bonds in which a pair of electrons common to two atoms is generally held equally by each of these two atoms (ie side chain is not polar). Genetically encoded non-polar amino acids include Gly, Leu, Val, Ile, Ala, and Met. Non-polar amino acids can be further separated to isolate aliphatic amino acids, which are hydrophobic amino acids with an aliphatic carbohydrate side chain. Genetically encoded aliphatic amino acids include Ala, Leu, Val and Ile.

[00139] A polar amino acid is a hydrophilic amino acid with a side chain that is uncharged at a physiological pH but which contains one bond in which a pair of electrons shared by two atoms is more closely held by one of the atoms. Genetically encoded polar amino acids include Ser, Thr, Asn, and Gln.

[00140] An acidic amino acid is a hydrophilic amino acid with a pKa of a side chain of less than 7. Acidic amino acids typically have negatively charged side chains at physiological pH due to a lack of a hydrogen ion. Genetically encoded acidic amino acids include Asp and Glu. The main amino acid is a hydrophilic amino acid with a pKa value of the side chain of more than 7. The main amino acids, as a rule, have positively charged side chains at a physiological pH level due to association with the hydronium ion. Genetically encoded basic amino acids include Arg, Lys and His.

[00141] The% identity of amino acid sequences is defined as the number of matching identical residues divided by the total number of residues of the "longer" sequence in the comparison window. A “longer” sequence is a sequence containing more actual balances in the comparison window (the gaps introduced by WU-Blast-2 to maximize the alignment score are ignored).

[00142] Alignment may include introducing gaps into sequences intended for alignment. In addition, it is understood that for sequences that contain more or less amino acids than the protein encoded by the sequence of the disclosed polypeptide, in one case, the percentage of sequence identity is determined based on the number of identical amino acids relative to the total number of amino acids. When calculating the percentage of identity, the various manifestations of sequence variability, such as insertions, deletions, substitutions, etc., do not ascribe the relative mass.

[00143] In one case, a positive value (+1) is assigned only to identical residues, and all forms of sequence variation, including gaps, are assigned the value “0”, which eliminates the need for a weighted scale or parameters, as described below to calculate sequence similarity. The percentage of sequence identity can be calculated, for example, by dividing the number of identical identical residues by the total number of residues of the “shorter” sequence in the aligned region and multiplying by 100. The “longer” sequence is a sequence containing more actual residues in the aligned region.

Wireframes

[00144] As noted herein, antibodies of the present invention may contain a framework structure into which the above described CDRs can be grafted. In one embodiment, the framework structure is a conventional antibody structure, which means that the antibody contains the variable domain sequences of two heavy and two light chains. In some cases, the antibody combinations described herein may contain additional components (frame region, J and D regions, constant regions, etc.) that make up the heavy and / or light chain. Some embodiments of the invention include the use of human framework components.

[00145] Accordingly, in various embodiments, the antibodies of the invention comprise frameworks of conventional antibodies. In some embodiments of the invention, the disclosed antibodies can be human and monoclonal antibodies, bispecific antibodies, diabodies, minuets, domain antibodies, synthetic antibodies, chimeric antibodies, fused antibodies, and fragments of each of these components, respectively. The above CDRs and combinations of CDRs can be grafted into any of the following scaffolds.

[00146] The chimeric antibodies of the present invention may comprise a heavy and / or light chain sequence identical or homologous to the corresponding sequences obtained from a particular species. For example, in one embodiment, the anti-Bb factor antibody is a chimeric antibody containing a human Fc domain, while the remainder of the antibody may be identical or homologous to the corresponding mouse or rodent sequences. Chimeric antibodies can represent fragments of such antibodies as long as these fragments show the necessary biological activity and contain the sequence obtained from another species, class of antibodies or a subclass of antibodies (US patent No. 4816567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81: 6851-6855).

[00147] In some embodiments of the invention, the variable region of the disclosed anti-Bb antibody contains at least three heavy or light chain CDRs, see above (Kabat et al., 1991, Sequences of Proteins of Immunological Interest, Public Health Service NIH, Bethesda, MD; see also Chothia and Lesk, 1987, J. Mol Biol 196: 901-917; Chothia et al., 1989, Nature 342: 877-883), within the frame region (frame regions are designated 1-4, FR1, FR2, FR3 and FR4 according to Kabat et al., 1991, supra; see also Chothia and Lesk, 1987, supra).

[00148] In some cases, the antibody may consist of the sequence of the variable domain of the heavy chain and the sequence of the variable domain of the light chain. In some cases, the sequence of the variable domain of the heavy or light chain may contain a sequence selected from the sequences from Table 3.

[00149] The structural units of a conventional antibody in most cases comprise a tetramer. Each tetramer, as a rule, consists of two identical pairs of polypeptide chains, with each pair containing one light chain (usually having a molecular weight of about 25 kDa) and one heavy chain (usually having a molecular weight of about 50-70 kDa) . The amino-terminal part of each chain contains a variable region of from about 100 to 110 or more amino acids, which are mainly responsible for antigen recognition. The carboxy-terminal part of each chain defines a constant region, and the heavy chain can contain only three constant regions (CH1, CH2 and CH3), while constant regions can contribute to the regulation of effector function. Human light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, gamma, alpha or epsilon and determine the antibody isotype, respectively, as IgM, IgD, IgG, IgA and IgE. IgG has several subclasses, including, but not limited to, IgG1, IgG2, IgG3 and IgG4. IgM has subclasses, including, but not limited to, IgM1 and IgM2.

[00150] In the light and heavy chains, the variable and constant regions are connected via a “J” region of about twelve (12) or more amino acids in length, and the heavy chain also contains a “D” region of about ten (10) or more amino acids in length. In general, see Paul, W., ed., 1989, Fundamental Immunology Ch. 7, 2nd ed. Raven Press, N.Y. The variable regions of each pair of light and heavy chains form the binding region of the antibody.

[00151] Some antibodies of natural origin, for example, which can be found in camels and llamas, are dimers consisting of two heavy chains, and do not include light chains. Muldermans et al., 2001, J. Biotechnol. 74: 277-302; Desmyter et al., 2001, J. Biol. Chem. 276: 26285-26290. Crystallographic studies of camelid antibodies revealed that the CDR3 regions form a surface that interacts with the antigen and, thus, is critical for antigen binding, as in more typical tetrameric antibodies. The invention includes dimeric antibodies consisting of two heavy chains, or fragments thereof, which can bind to and / or inhibit the biological activity of factor Bb.

[00152] The variable regions of the heavy and light chains, as a rule, have the same overall structure from relatively conservative framework regions (FR) connected by three complementarity determining regions or CDRs. CDRs contain hypervariable regions of an antibody that are responsible for antigen recognition and binding. CDRs of two strands of each pair are oriented and supported by frame regions, which makes it possible to bind a particular epitope. From the N-terminus to the C-terminus, both heavy and light chains contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is carried out in accordance with the definitions from the Kabat Sequences of Proteins of Immunological Interest. Chothia et al., 1987, J. Mol. Biol. 196: 901-917; Chothia et al., 1989. Nature 342: 878-883.

[00153] CDRs constitute the main surface contact points for antigen binding. See, for example, Chothia and Lesk, 1987, J. Mol. Biol. 196: 901-917. In addition, light chain CDR3 and in particular heavy chain CDR3 may constitute the most important determinants of antigen binding in the variable regions of the light and heavy chains. See, for example, Chothia and Lesk, 1987, supra; Desiderio et al., 2001, J. Mol Biol 310: 603-615; Xu and Davis, 2000, Immunity 13: 37-45; Desmyter et al., 2001, J. Biol. Chem. 276: 26285-26290; and Muyldermans, 2001, J. Biotechnol. 74: 277-302. In some antibodies, the heavy chain CDR3 constitutes the main contact area between the antigen and the antibody. Desmyter et al., 2001, supra. In vitro selection schemes in which only CDR3 is varied can be used to vary the binding properties of the antibody. Muyldermans, 2001, supra; Desiderio et al., 2001, supra.

[00154] Antibodies of natural origin typically contain a signal sequence that directs the antibody into the cell pathway for protein secretion and which is not present in the mature antibody. A polynucleotide encoding an antibody of the invention can encode a signal sequence of natural origin or a heterologous signal sequence, as described below.

[00155] In one embodiment, the anti-Bb factor antibody is a monoclonal antibody with from one (1) to six (6) CDRs, as described herein. The antibodies of the invention can be of any type, including IgM, IgG (including IgG1, IgG2, IgG3, IgG4), IgD, IgA, or IgE antibodies. In a specific embodiment, the antibody is an IgG type antibody. In a more specific embodiment, the antibody is an IgG2 type antibody.

[00156] In some embodiments of the invention, the antibody may contain complete heavy and light chains with all CDRs from one species, for example, a human. In an alternative embodiment, for example, in embodiments of the invention in which the antibody contains less than six CDRs from the above sequences, additional CDRs may belong to a different species (e.g., murine CDRs), or they may be human CDRs that are different from those shown in the sequences. For example, human regions of HC CDR3 and LC CDR3 from the corresponding sequences defined herein can be used with HC CDR1, HC CDR2, LC CDR1 and LC CDR2, optionally selected from other species or from other human antibody sequences, or combinations thereof. For example, the CDRs of the invention can replace CDR regions of commercially relevant chimeric or humanized antibodies.

[00157] In specific embodiments of the invention, antibody framework components that are human components are used.

[00158] In some embodiments of the invention, however, the frame components may be a mixture of different species. Therefore, the antibody may be a chimeric antibody and / or a humanized antibody. In general, both chimeric and humanized antibodies can be antibodies in which regions or amino acids from more than one species are combined. For example, chimeric antibodies in most embodiments of the invention comprise variable region (s) from a mouse, rat, rabbit or other suitable non-human animal and constant region (s) from a human.

[00159] Humanized antibodies are antibodies that are originally derived from non-human antibodies, for example, a murine antibody. In various embodiments of a humanized anti-Bb factor antibody, framework regions of the variable domain or framework amino acids that are derived from a non-human antibody can be replaced with identical amino acids located at corresponding positions in human antibodies. In some embodiments of a humanized antibody, an entire antibody with the exception of CDRs may be encoded by a polynucleotide of human origin or identical to such an antibody with the exception of CDR regions. In other embodiments, a humanized antibody may contain specific amino acid positions in which residues have been replaced by residues of the same or similar position in the corresponding human antibody. CDRs, some or all of which can be encoded by nucleic acids belonging to a nonhuman organism, are inoculated into the beta-folded framework of the variable region of a human antibody to create an antibody whose specificity is determined by the grafted CDRs. The creation of such antibodies is described, for example, in WO 92/11018, Jones, 1986, Nature 321: 522-525, Verhoeyen et al., 1988, Science 239: 1534-1536. Humanized antibodies can also be obtained using mice with a genetically engineered immune system. Roque et al., 2004, Biotechnol Prog. 20: 639-654. In some embodiments, the CDRs may be human, and thus, in this context, both humanized and chimeric antibodies contain some non-human CDRs. In some cases, it is possible to obtain humanized antibodies that contain HC CDR3 and LC CDR3 regions from one or more other CDR regions of different species origin.

[00160] In one embodiment, the anti-Bb factor antibody may be a multispecific antibody, namely, a bispecific antibody (eg, a diatel). These are antibodies that bind to two (or more) different antigens, for example, factor Bb and another antigen, or two different epitopes of factor Bb. Diabodies can be obtained by a variety of methods known in the art (Holliger and Winter, 1993, Current Opinion Biotechnol. 4: 446-449), for example, obtained chemically or from hybrid hybridomas.

[00161] In one embodiment, the anti-Bb factor antibody is a minitool. Minobodies are minimized antibody-like proteins containing scFv linked to the CH3 domain. Hu et al., 1996, Cancer Res. 56: 3055-3061.

[00162] In one embodiment, the anti-Bb factor antibody is a domain antibody; see, for example, US patent No. 6248516. Domain antibodies (dAb) are functional antibody binding domains corresponding to the variable regions of the heavy (VH) or light (VL) chains of human antibodies. dABs have a molecular weight of approximately 13 kDa or less than one tenth of the total antibody size. dABs are well expressed in a variety of hosts, including bacterial, yeast, and mammalian cell systems. In addition, dAb are highly stable and remain active even after exposure to harsh conditions such as lyophilization or thermal denaturation. See, for example, US patent 6291158; 6,582,915; 6,593,081; 6172197; USA Serial No. 2004/0110941; European patent 0368684; US patent 6696245, WO 04/058821, WO 04/003019 and WO 03/002609, all of which are fully incorporated by reference.

[00163] In one embodiment, an anti-Bb factor antibody is a fragment of an antibody that is a fragment of any of the antibodies described herein that retains binding specificity for factor Bb. In various embodiments, the antibodies are F (ab), F (ab '), F (ab') ₂ , Fv fragments, or Fv single chain fragments. In the context of this document, the antibody at least contains a polypeptide that can specifically bind to an antigen, wherein the polypeptide contains all or part of the variable region of the heavy and / or light chain.

[00164] Specific antibody fragments include, but are not limited to, (i) a Fab fragment consisting of VL, VH, CL, and CH1 domains, (ii) an Fd fragment consisting of VH and CH1 domains, (iii) an Fv fragment consisting of from the VL and VH domains of a single antibody; (iv) a dAb fragment (Ward et al., 1989, Nature 341: 544-546), which consists of one variable region, (v) isolated CDR regions, (vi) a F (ab ') fragment ₂ , a bivalent fragment containing two linked Fab (vii) fragments of single-chain Fv (scFv) molecules in which the VH domain and the VL domain are linked by a peptide linker that allows the association of two domains to form an antigen-binding site (Bird et al., 1988, Science 242: 423-426, Huston et al., 1988, Proc. Natl Acad. Sci. USA 85: 5879-5883), (viii) bispecific single chain Fv dimers (PCT / US92 / 09965) and (ix) of a die or triatel, multivalent or multispecific fragments constructed by gene fusion (Tomlinson et. al, 2000, Methods Enzymol. 326: 461-479; WO 94/13804; Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90: 6444-6448 ) Antibody fragments may be modified. For example, molecules can be stabilized by incorporating disulfide bridges linking the VH and VL domains (Reiter et al., 1996, Nature Biotech. 14: 1239-1245). Again, as indicated herein, non-CDR components of these fragments are preferably represented by human sequences.

[00165] In one embodiment, the anti-Bb factor antibody is a conventional antibody, for example, a human immunoglobulin. In this embodiment, as indicated above, the specific structures comprise complete heavy and light chains containing CDR regions. In further embodiments, one or more CDRs of the invention are used with other CDRs, framework regions, J and D regions, constant regions, etc., belonging to other human antibodies. For example, the CDRs of the invention can replace the CDRs of any number of human antibodies, in particular, commercially relevant antibodies.

[00166] In one embodiment, the anti-Bb factor antibody is an antibody fusion protein (eg, an antibody conjugate). In this embodiment, the antibody is fused to a conjugation partner. The conjugation partner may be protein or non-protein; the latter is generally obtained using functional groups on an antibody (see discussion of covalent modifications of antibodies) and on a conjugation partner. For example, linkers are known in the art; for example, homo- or hetero-bifunctional linkers are well known (see Pierce Chemical Company catalog, cross-linker technical section, pages 155-200, incorporated herein by reference).

[00167] In one embodiment, the anti-Bb factor antibody is an analogue of an antibody. In some cases, antibody analogs may be called synthetic antibodies. For example, many recent works use alternative protein scaffolds or artificial CDR grafted scaffolds. Such scaffolds include, but are not limited to, mutations introduced to stabilize the three-dimensional structure of the antibody, as well as fully synthetic scaffolds consisting, for example, of biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1: 121-129. Roque et al., 2004, Biotechnol. Prog. 20: 639-654. In addition, you can use peptide antibody mimetics (PMA), as well as work on the basis of antibody mimetics using fibronectin components as a framework.

VH and VL options

[00168] As indicated above, in some embodiments, the invention provides antibodies comprising or consisting of a heavy chain variable region comprising SEQ ID NO: 1-3 and / or a light chain variable region comprising SEQ ID NO: 4-6 , respectively, or fragments thereof as defined above. Thus, in these embodiments of the invention, the antibody contains not only at least one CDR or variant, but also at least a portion of the frame sequence. In addition, the invention includes variants of such heavy chain variable sequences or light chain variable sequences.

[00169] A variant variable region generally has an amino acid homology, similarity or identity of at least 80% to a parent variable region, such as the regions disclosed herein. In some embodiments, the homology or identity of the variant and parental sequences is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% and almost 100%. The homology, similarity or identity of the nucleotide sequences between the nucleotide sequences encoding the individual variant VH and VL and the nucleotide sequences provided herein is at least 70%, and generally with preferably increasing homology or identity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and almost 100%. In addition, the variant variable region may, in many embodiments, have a biological function, including but not limited to 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the specificity and / or activity of the parental CDR. In some cases, homology and / or identity is determined only outside of the CDR sequences, which may be identical. In other cases, homology and / or identity is determined over the entire length of the sequence, including CDR sequences. In some embodiments, constant region variants may also be included.

[00170] In various cases, the homology of amino acid sequences can display a percentage of identity or positive results with optimal alignment, as described above. In various cases,% homology (% positive results) or% identity can be calculated by dividing the number of aligned amino acids in the comparison window. The comparison window may be the full length of one or the other polypeptide, if the length of the two polypeptides is not the same. In other cases, the comparison window may form part of one of the polypeptides. In various cases, the comparison window for determining the homology or identity of two polypeptide sequences is more than about 40 ak (amino acids), 45 ak, 50 ak, 55 ak, 60 ak, 65 ak, 70 ak, 75 ak, 80 ak, 85 ak. 90 ak, 95 ak, 100 ak, 150 ak or 200 ak, and / or less than about 200 ak, 150 ak, 100 ak, 95 ak, 90 ak, 85 ak, 80 ak, 75 ak, 70 ak, 65 ak 60 ak, 55 ak, 50 ak or 45 ak. In some embodiments of the invention, as is the case with CDR sequences, the comparison window may be less than 40 ak, for example, between less than about 25 ak, 24 ak, 23 ak, 22 ak, 21 ak, 20 ak, 19 ak, 18 ak, 17 ak, 16 ak, 15 ak, 14 ak, 13 ak, 12 ak, 11 ak, 10 ak, 9 ak, 8 ak, 7 ak, 6 ak, 5 ak or 4 ak, and more than about 3 ak , 4 ak, 5 ak, 6 ak, 7 ak, 8 ak, 9 ak, 10 ak, 11 ak, 12 ak, 13 ak, 14 ak, 15 ak, 16 ak, 17 ak, 18 ak, 19 ak, 20 ak, 21 ak, 22 ak, 23 ak or 24 ak.

[00171] In various cases, the claimed amino acid sequences may have% identity or% homology (% positive) greater than a predetermined comparison window, i.e. greater than about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and / or less than about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80% or 75%.

Covalent modifications of antibodies to factor Bb

[00172] Covalent modifications of antibodies are included within the scope of this invention and generally, but not always, occur after translation. For example, several types of covalent modifications of an antibody are introduced into a molecule by reacting specific amino acid residues of an antibody with an organic derivatizing agent that is capable of reacting with selected side chains of the N- or C-terminal residues.

[00173] Cysteinyl residues are most often reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. The cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β- (5-imidozoyl) propionic acid, chloroacetyl phosphate, N-alkyl maleimides, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, p-chloro-2-chloro-2-chloro -4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[00174] Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5-7.0, since this agent is relatively specific for the histidyl side chain. Also applicable is para-bromophenacyl bromide; the reaction is preferably carried out in a 0.1 M sodium cacodilate solution at pH 6.0.

[00175] Lysinyl and amino terminal residues are reacted with succinic anhydride or anhydrides of other carboxylic acids. Derivatization by these agents can lead to a change in the charge of lysinyl residues. Other suitable reagents for derivatizing alpha-amino residues include imido esters, such as methyl picolinimidate; pyridoxalphosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with glyoxylate.

[00176] Arginyl residues are modified by reaction with one or more conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and ninhydrin. To derivatize arginine residues, it is necessary that the reaction be carried out in an alkaline medium due to the high pK _{a of the} guanidine functional group. In addition, such reagents can interact with lysine groups, as well as with the epsilon-amino group of arginine.

[00177] It is possible to carry out specific modifications of tyrosyl residues, the introduction of spectral labels into tyrosyl residues by reaction with aromatic compounds of diazonium or tetranitromethane is particularly interesting. The most commonly used are N-acetylimidisole and tetranitromethane to form O-acetyl tyrosyl and 3-nitro derivatives, respectively. Tyrosyl residues are iodinated using ¹²⁵ I or ¹³¹ I to produce labeled proteins for use in radioimmunoassay, with the chloramine T method described above being suitable.

[00178] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N = C = N-R '), where R and R' are optionally different alkyl groups, such as 1-cyclohexyl -3- (2-morpholinyl-4-ethyl) carbodiimide or 1-ethyl-3- (4-azonia-4,4-dimethylphenyl) carbodiimide. In addition, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[00179] Derivatization with bifunctional agents is used to crosslink antibodies with a water-insoluble matrix substrate or surface for use in various methods. Commonly used cross-linking agents include, for example, 1,1-bis (diazoacetyl) -2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imido esters, including disuccinimidyl esters, such as 3 '-dithio-bis- (succinimidyl propionate), and bifunctional maleimides, such as bis-N-maleimido-1,8-octane. Using derivatizing agents such as methyl 3 - [(p-azidophenyl) dithio] propioimidate, photoactivated intermediates are obtained which are capable of cross-linking in the presence of light. Alternatively, water-insoluble matrices, such as cyanogen bromide-activated carbohydrates and reactive substrates described in US Pat. No. 3,969,287; are used to immobilize the protein; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4330440.

[00180] Glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Any form of these residues is included in the scope of the present invention.

[00181] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (ie, Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of an N-terminal amine and amidation of any C-terminal carboxyl group.

Glycosylation

[00182] Another type of covalent modification of antibodies, included in the scope of this invention, includes changing the profile of protein glycosylation. As is known in the art, glycosylation profiles can depend on both the protein sequence (for example, the presence or absence of specific glycosylation amino acid residues discussed below) and the host cell or host organism in which the protein is produced. Specific expression systems are discussed below.

[00183] Glycosylation of polypeptides is typically N-linked or O-linked. N-linked glycosylation refers to the attachment of a carbohydrate component to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, represent recognition sequences for the enzymatic attachment of the carbohydrate component to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences creates a potential glycosylation site. O-linked glycosylation refers to the addition of one of the sugars of N-acetylgalactosamine, galactose or xylose to a hydroxy amino acid, most often serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[00184] Adding glycosylation sites to the disclosed antibody is conveniently done by changing the amino acid sequence so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). The change can also be carried out by adding or substituting one or more serine or threonine residues in the original sequence (for O-linked glycosylation sites). For convenience, the amino acid sequence of the antibody is preferably changed at the DNA level, in particular by mutating the DNA encoding the target polypeptide in preselected bases so that the generated codons are translated into the necessary amino acids.

[00185] Another way to increase the number of carbohydrate components in an antibody is by chemical or enzymatic conjugation of glycosides with a protein. These procedures are advantageous since they do not require the production of protein in the host cell, which has the ability to carry out N- or O-linked glycosylation. Depending on the conjugation method used, sugar (a) can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as cysteine groups, (d) free hydroxyl groups, such as serine, threonine or hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine or tryptophan residues, or (f) the amide group of glutamine. These methods are described in WO 87/05330, published September 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., Pp. 259-306.

[00186] Removing the carbohydrate components present on the parent antibody can be carried out chemically or enzymatically. For chemical deglycosylation, it is necessary to expose the protein to the action of a trifluoromethanesulfonic acid compound or an equivalent compound. This treatment results in the breakdown of most or all of the sugars, except the binding sugar (N-acetylglucosamine or N-acetylgalactosamine), while the polypeptide remains intact. Chemical deglycosylation is described in Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259: 52 and by Edge et al., 1981, Anal. Biochem. 118: 131. Enzymatic cleavage of carbohydrate components on a polypeptide can be carried out using various endo- and exoglycosidases, as described in Thotakura et al., 1987, Meth. Enzymol 138: 350. Glycosylation at potential glycosylation sites can be prevented by using the tunicamycin compound as described in Duskin et al., 1982, J. Biol. Chem. 257: 3105. Tunicamycin blocks the formation of protein-N-glycoside bonds.

Pegylation

[00187] Another type of covalent modification of an antibody includes the attachment of various non-protein polymers to the antibody, including, but not limited to, various polyols, such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, as described in US Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 and / or 4,179,337, all of which are fully incorporated by reference. In addition, as is known in the art, amino acid substitutions can be made at various antibody positions to facilitate the addition of polymers such as PEG.

Tags

[00188] In some embodiments of the invention, the covalent modification of antibodies according to the invention includes the addition of one or more labels.

[00189] The term "labeling group" refers to any detectable label. Examples of suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g. ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I), fluorescent groups ( for example, FITC, rhodamine, phosphors based on lanthanide complexes), enzymatic groups (for example, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups or predefined polypeptide epitopes recognized by, for example, secondary reporter pairs lightning binding of secondary antibodies, metal-binding domains, epitope labels). In some embodiments of the invention, the labeling group is coupled to the antibody via spacer legs of various lengths to reduce potential steric mismatch. Various methods for labeling proteins that can be used to carry out the present invention are known in the art.

[00190] In general, labels are divided into different classes depending on the method for their identification: a) isotopic labels, which may be radioactive or heavy isotopes; b) magnetic marks (e.g. magnetic particles); c) redox active components; d) optical dyes; enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predefined polypeptide epitopes recognized by the secondary reporter (for example, paired leucine zipper sequences, binding sites of secondary antibodies, metal binding domains, epitope tags, etc.). In some embodiments of the invention, the labeling group is coupled to the antibody via spacer legs of various lengths to reduce potential steric mismatch. Various methods for labeling proteins that can be used to carry out the present invention are known in the art.

[00191] Specific labels include optical dyes, including but not limited to chromophores, phosphors, and fluorophores, the latter being specific in various cases. Fluorophores can be both “low molecular weight” fluorophores and protein fluorophores.

[00192] A fluorescent label can be any molecule that can be detected due to its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrodamine, eosin, erythrosine, coumarin, methylcoumarins, pyrene, malachite green, stilbene, yellow lucifer, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), cascade blue, cascade yellow and R-phycoerythrin (FE) (Molecular Probes, Eugene, OR), FITC, rhodamine and Texas red (Pierce, Rockford, IL), Cy5, Cy5.5, Cy7 ( Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores, are described in Molecular Probes Handbook by Richard P. Haugland, expressly incorporated herein by reference.

[00193] Suitable protein fluorescent labels also include, but are not limited to, green fluorescent protein, including PBS species Renilla, Ptilosarcus or Aequorea (Chalfie et al., 1994, Science 263: 802-805), UZFP (Clontech Laboratories, Inc. , Genbank Accession Number U55762), Blue Fluorescent Protein (HFB, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24: 462-471; Heim et al ., 1996, Curr. Biol 6: 178-182), amplified yellow fluorescent protein (UVBF, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol 150: 5408-5417), β-galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. USA 85: 2603-2607) and Renilla (WO 92/15673, WO 95/07463, WO 98/14605, WO 9 8/26277, WO 99/49019, US Pat.

Polynucleotides Encoding Antibodies Against Factor Bb

[00194] In certain aspects, the invention provides nucleic acid molecules encoding the antibodies described herein. In some cases, the disclosed nucleic acids encode IgG, variable regions, or CDRs described herein. Nucleic acids include both DNA and RNA molecules. Nucleic amino acids can be nucleic acids of both natural and synthetic origin. The nucleic acids of the present invention are typically polynucleic acids; which means polymers from single nucleotides that are covalently linked via 3 ', 5' phosphodiester bonds. In various cases, polynucleotide sequences may be single-stranded, double-stranded, or combined. In some variations, the nucleotide sequences may contain natural nucleic acids, synthetic nucleic acids, unnatural nucleic acids and / or nucleic acid analogs. Nucleotide sequences may further comprise molecules other than nucleic acids, such as amino acids and other monomers.

[00195] In many embodiments, the coding sequence may be an isolated nucleic acid molecule. An isolated nucleic acid molecule is detected and separated from at least one component with which it is usually associated in a natural source. In some cases, this component may be a nucleotide sequence, a protein, or a non-protein molecule. An isolated nucleic acid molecule encoding an antibody against factor Bb differs from the form in which it is found in nature. Therefore, the isolated nucleic acid molecules encoding antibodies against factor Bb are different from the encoding nucleic acid molecules (s) present in natural cells. Moreover, the isolated nucleic acid molecule encoding an anti-factor Bb antibody includes nucleic acid molecules encoding anti-factor Bb antibodies contained in cells that typically express an anti-factor Bb antibody, where, for example, the nucleic acid molecule is in a chromosome location that is different from locations in natural cells. Therefore, the isolated nucleic acid molecules are different from the nucleic acid molecule present in the body. Moreover, in some cases, the isolated nucleic acid molecule may be the nucleic acid contained in the cell, for example, if the selected nucleic acid molecule was introduced into the cell and it is in an extrachromosomal location or in a chromosomal location other than its native location.

[00196] Depending on the application, the nucleic acid may be double-stranded, single-stranded, or contain portions of both double-stranded and single-stranded sequences. As understood by those skilled in the art, the description of one chain ("Watson") also determines the sequence of another chain ("Scream"). A recombinant nucleic acid may be an amino acid originally formed in vitro, generally by treating the nucleic acid with endonucleases in a form that is not normally found in nature. Thus, an isolated antibody can be encoded in a linear form by a nucleic acid or an expression vector formed in vitro by ligation of DNA molecules that are usually not connected, and in both cases is considered recombinant for the purposes of this invention. It is clear that if a recombinant nucleic acid with all the necessary regulatory elements is obtained and introduced into the host organism, it can replicate non-recombinantly, i.e. using in vivo cell apparatus of the host cell, and not in vitro manipulations; however, such nucleic acids after recombinant production, although subsequently non-recombinantly replicated, are still considered recombinant for the purposes of this invention.

[00197] In some embodiments of the invention, the recombinant nucleic acid may contain one or more regulatory elements or regulatory sequences. Regulatory elements and regulatory sequences refer to nucleotide sequences necessary for the expression of a functionally linked coding sequence in a particular host organism. Regulatory sequences that are suitable for prokaryotes, for example, include a promoter, an operator sequence, and a ribosome binding site. In eukaryotic cells, promoters, polyadenylation signals, and enhancers are known to be used. In the context of this document, a functionally linked sequence is a nucleotide sequence that is in functional relationship with another nucleotide sequence. For example, coding nucleotide sequences may be operably linked to regulatory nucleotide sequences. For example, DNA for a subsequence or secretory leader sequence is operably linked to DNA for a polypeptide if it is expressed as a precursor protein that is involved in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. In most embodiments of the invention, the functionally linked sequence is a DNA sequence covalently linked, for example, to a secretory leader sequence. In many embodiments of the invention, it is not necessary that the enhancer sequences are adjacent to the coding sequence; on the contrary, the two sequences can be separated by one or more nucleic acids.

[00198] In various cases, the nucleic acids of the disclosed nucleotide sequences may contain nucleotides that are metabolized in a similar manner to nucleotides of natural origin. Also included are nucleic acid-like structures with synthetic skeleton analogs, including, without limitation, phosphodiester, thiophosphate, dithiophosphate, methylphosphonate, phosphoramidate, alkyl phosphodiester, sulfamate, 3'-thioacetal, methylene (methylimino), 3'-N-carbamate and peptide nucleic acids (PNAs) (see, for example: "Oligonucleotides and Analogues, and Practical Approach," edited by F. Eckstein, IRL Press at Oxford University Press (1991); "Antisense Strategies," Annals of the New York Academy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992); Milligan (1993) J. Med. Chem. 36: 1923-1937; and "Antisense Research and Applications "(1993, CRC Press)). PNAs contain non-ionic skeletons, such as N- (2-aminoethyl) glycine units. Thiophosphate bonds are described in: WO 97/03211; WO 96/39154; and Mata (1997) Toxicol. Appl. Pharmacol 144: 189-197. Other synthetic skeletons falling within this term include methylphosphonate bonds or alternating methylphosphonate and phosphodiester bonds (Strauss-Soukup (1997) Biochemistry 36: 8692-8698) and benzylphosphonate bonds (Samstag (1996) Antisense Nucleic Acid Drug Dev 6: 153-156 )

[00199] As those skilled in the art understand, due to the degeneracy of the genetic code, a much larger number of nucleic acids can be obtained that all encode the CDRs (as well as heavy and light chains or other components of an antibody) according to the present invention. Thus, after determining a specific amino acid sequence, those skilled in the art can obtain any number of different nucleic acids by simply modifying the sequence of one or more codons so as not to alter the amino acid sequence of the encoded protein.

[00200] In various cases, nucleotide sequences encoding the polypeptide sequences of SEQ ID NO: 1-6 and 8-15, and 18-31 are included. These coding nucleotide sequences can be translated into a polypeptide having an amino acid sequence identical to the disclosed polypeptide sequence. In many cases, nucleotides encoding identical polypeptides may not have identical nucleotide sequences. This is due to the degeneracy of the genetic code. The disclosed coding sequences may further comprise untranslated sequences, for example, polyadenylation sequences. The coding sequences of the invention may also contain an intron or an intermediate untranslated sequence that are excised from the transcribed mRNA before translation. In various cases, the transcribed mRNA can be capped with terminal 7-methylguanosine. In some embodiments of the invention, the coding sequences include coding sequences of amino acids that are not present in the final antibody, for example, sequences necessary for exporting the antibody.

[00201] In some variations, due to the degeneracy of the genetic code, multiple coding nucleotide sequences can encode a single polypeptide sequence. Such coding nucleotide sequences according to the invention can also be homologous to nucleotide sequences that encode the disclosed polypeptides. Alignment of the coding nucleotide sequences can be carried out using BLASTn, as described above. In various cases, the homology (or identity in BLASTn) of these aligned nucleotide sequences can be more than about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90 % or 95% and / or less than about 100%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50% or 45%. In various cases, aligned homologous sequences may be less than about 700 nucleotides, 600 nucleotides, 500 nucleotides, 400 nucleotides, 300 nucleotides, 200 nucleotides, 100 nucleotides, 90 nucleotides, 80 nucleotides, 70 nucleotides, 60 nucleotides, 50 nucleotides or 40 nucleotides and / or more than about 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides or 600 nucleotides.

[00202] In various cases, the coding sequence controls the transcription of the ribonucleotide sequence, which can be translated into an amino acid sequence in accordance with the standard genetic code. In various cases, the code may include variations of the canonical code. In some variations, the coding sequence may contain introns or intermediate sequences that do not encode amino acids, but can be transcribed and subsequently deleted before translation of the ribonucleic acid into the polypeptide.

Methods for producing antibodies

[00203] The present invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcriptional or expression cassettes that contain at least one of the above polynucleotides. In addition, the invention provides host cells containing such expression constructs.

[00204] Typically, expression vectors used in any host cell contain sequences for maintaining plasmids and for cloning and expression of exogenous nucleotide sequences. Such sequences, collectively referred to as flanking sequences, in certain embodiments of the invention typically comprise one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, a replication start point, a transcription termination sequence, a complete intron sequence containing a donor and an acceptor splicing sites, a sequence encoding a leader sequence for the secretion of the polypeptide, the binding site Ia ribosomes, polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding a polypeptide intended for expression and a selectable marker element. Each of these sequences is discussed below.

[00205] Optionally, the vector may contain a sequence encoding a "label", ie an oligonucleotide molecule located at the 5 'or 3' end of the coding sequence of an antibody against Bb factor; the oligonucleotide sequence can encode a polyHis tag (such as hexaHis) or another "tag" such as FLAG, HA (influenza virus hemagglutinin) or myc, for which commercially available antibodies exist. Such a label usually fuses with the polypeptide after expression of the polypeptide and can serve as a means for affinity purification or detection of antibodies to Bb factor in the host cell. Affinity purification can be performed, for example, by column chromatography using anti-tag antibodies as an affinity matrix. Optionally, the label can subsequently be removed from the purified anti-Bb factor antibody in various ways, such as using certain peptidases for cleavage.

[00206] Flanking sequences can be homologous (ie, belong to the same species and / or strain as the host cell), heterologous (ie, belong to a species different from the species or strain of the host cell), hybrid ( i.e. a combination of flanking sequences from more than one source), synthetic or native. Therefore, the source of the flanking sequence can be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional and can be activated by the apparatus of the host cell.

[00207] Flanking sequences used in the vectors of this invention can be obtained by any of several methods well known in the art. Typically, the flanking sequences used herein have been previously identified by mapping and / or digestion with restriction endonucleases and, therefore, can be isolated from the corresponding tissue source using suitable restriction endonucleases. In some cases, the complete nucleotide sequence of the flanking sequence may be known. Or the flanking sequence can be synthesized using the methods described herein for the synthesis and cloning of nucleic acids.

[00208] Regardless of whether all or only part of the flanking sequence is known, it can be obtained by polymerase chain reaction (PCR) and / or by screening the genomic library with a suitable probe, such as an oligonucleotide and / or fragment of the flanking sequence, from the same or other kind. If the flanking sequence is unknown, a DNA fragment containing the flanking sequence can be isolated from a larger portion of the DNA, which may contain, for example, a coding sequence and even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, column chromatography, Qiagen ^® (Chatsworth, CA), or other methods known to those skilled in the art. The selection of suitable enzymes for this purpose is obvious to a person skilled in the art.

[00209] The origin of replication is typically part of commercially acquired prokaryotic expression vectors and promotes amplification of the vector in the host cell. If the selected vector does not contain a replication start point, it can be chemically synthesized based on a known sequence and ligated into a vector. For example, the origin of replication from plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most gram-negative bacteria, and various viral sources (e.g. SV40, polyomas, adenovirus, vesicular stomatitis virus (BBC), or human papillomaviruses such as HPV or CMV) ) are suitable for cloning vectors in mammalian cells. In general, the component of the origin of replication is optional in mammalian expression vectors (for example, the SV40 point is often used only because it also contains an early viral promoter).

[00210] The transcription termination sequence is usually located 3 'to the end of the polypeptide-coding region and serves to terminate the transcription. Typically, the transcription termination sequence in prokaryotic cells is a G-C-rich fragment, followed by a poly-T sequence. Although the sequence is easy to clone from the library or even commercially available as part of a vector, it is also easy to synthesize using methods for the synthesis of nucleic acids, such as those described in this document.

[00211] The selectable marker gene encodes a protein necessary for the survival and growth of the host cell in a selective culture medium. Typical selectable marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, for example, ampicillin, tetracycline or kanamycin in the case of prokaryotic host cells; (b) make up for the auxotrophic deficiency in the cell; or (c) provide essential nutrients inaccessible from a complex or defined environment. Specific selective markers are the kanamycin resistance gene, ampicillin resistance gene, glutamine synthetase (GS), and the tetracycline resistance gene. Preferably, the neomycin resistance gene can also be used for selection in both prokaryotic and eukaryotic host cells.

[00212] Other amplified genes can be used to amplify a gene intended for expression. Amplification is a process in which the genes necessary for the production of a protein critical for cell growth or survival are reiterated in tandem on the chromosomes of subsequent generations of recombinant cells. Examples of suitable selective markers for mammalian cells include the dihydrofolate reductase gene (DHFR) and the non-promoter thymidine kinase gene. Mammalian cell transformants are placed under selection pressure conditions in which only the transformants are uniquely capable of survival due to the selective gene present in the vector. Selection pressure is created by culturing the transformed cells under conditions in which the concentration of the selection agent in the medium is successively increased, thereby amplifying both the selective gene and the DNA that encodes another gene, such as an antibody gene that binds to the factor Bb polypeptide or epitope of factor Bb. As a result, an increased amount of a polypeptide, such as an antibody against Bb factor, is synthesized from amplified DNA.

[00213] The ribosome binding site is usually necessary to initiate translation of mRNA and is characterized by the presence of a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes). This element is usually located 3 'to the promoter and 5' to the coding sequence for expression of the polypeptide.

[00214] In some cases, for example, when glycosylation is required in the expression system of a eukaryotic host cell, various pre- or pro-sequences can be manipulated to improve glycosylation or yield. For example, you can change the site of peptidase cleavage of a particular signal peptide or add pro-sequences that can also affect glycosylation. The final protein product may contain at position -1 (relative to the first amino acid of the mature protein) one or more additional amino acids associated with expression, which may not have been completely removed. For example, the final protein product may contain one or more amino acid residues located in the peptidase cleavage site attached to the amino terminus. Alternatively, the use of certain cleavage sites may result in a slightly truncated form of the desired polypeptide if the enzyme makes an incision in such a region of the mature polypeptide.

[00215] The expression and cloning vectors of the invention typically comprise a promoter that is recognized by the host organism and is operably linked to a molecule encoding an antibody against Bb factor. Promoters are non-transcribed sequences located above (i.e., 5 ′) the start codon of a structural gene (generally ranging from about 100 to 1000 bp) that regulate the transcription of the structural gene. Traditionally, promoters are divided into two classes: inducible promoters and constitutive promoters. Inducible promoters initiate elevated levels of transcription from DNA under their control in response to some changes in culture conditions, such as the presence or absence of nutrients or a change in temperature. On the other hand, constitutive promoters uniformly transcribe the gene with which they are functionally linked, which means weak or absent regulation of gene expression. A large number of promoters recognized by various potential host cells are well known. A suitable promoter is operably linked to DNA encoding the heavy chain or light chain belonging to the Bb factor antibody of the invention by removing the promoter from the DNA source by digestion with restriction enzymes and inserting the desired promoter sequence into the vector.

[00216] In some embodiments of the invention, yeast cells can be used to produce the antibodies against Bb factor disclosed herein. Also promoters suitable for use with yeast hosts are well known in the art. Yeast enhancers are preferably used with yeast promoters. Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, derived from virus genomes such as polyoma virus, chicken pox virus, adenovirus (such as adenovirus 2), cattle papilloma virus, avian sarcoma virus , cytomegalovirus, retroviruses, hepatitis B virus and, most preferably, monkey virus 40 (SV40). Other suitable mammalian promoters include heterologous mammalian promoters, for example, heat shock promoters and the actin promoter.

[00217] Additional promoters that may be of interest include, but are not limited to: the early SV40 promoter (Benoist and Chambon, 1981, Nature 290: 304-310); CMV promoter (Thornsen et al., 1984, Proc. Natl. Acad. U.S.A. 81: 659-663); a promoter contained in a 3 ′ long terminal repeat of Routh sarcoma virus (Yamamoto et al., 1980, Cell 22: 787-797); herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1444-1445); promoter and regulatory sequences from the metallothionine gene (Prinster et al., 1982, Nature 296: 39-42); and prokaryotic promoters, such as the beta-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75: 3727-3731); or a tac promoter (DeBoer et al., 1983, Proc. Natl Acad. Sci. U.S.A. 80: 21-25). The following transcriptional regulatory regions of animals that demonstrate tissue specificity and have been used in transgenic animals are also of interest: the regulatory region of the elastase I gene, active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald, 1987, Hepatology 7: 425-515); the regulatory region of the insulin gene active in pancreatic beta cells (Hanahan, 1985, Nature 315: 115-122); immunoglobulin gene regulatory region active in lymphoid cells (Grosschedl et al., 1984, Cell 38: 647-658; Adames et al., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol. Cell. Biol 7: 1436-1444); the regulatory region of the mouse mammary tumor virus active in the cells of the testes, mammary gland, lymphoid and mast cells (Leder et al., 1986, Cell 45: 485-495); the regulatory region of the albumin gene active in the liver (Pinkert et al., 1987, Genes and Devel. 1: 268-276); alpha-fetoprotein gene regulatory region active in the liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5: 1639-1648; Hammer et al., 1987, Science 253: 53-58); alpha-1-antitrypsin gene regulatory region active in the liver (Kelsey et al., 1987, Genes and Devel 1: 161-171); beta globin gene regulatory region active in myeloid cells (Mogram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-94); the regulatory region of the gene for myelin basic protein, active in the cells of the brain oligodendrocytes (Readhead et al., 1987, Cell 48: 703-712); the regulatory region of the myosin light chain-2 gene active in skeletal muscle (Sani, 1985, Nature 314: 283-286); and the regulatory region of the gonadotropic releasing hormone gene active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378).

[00218] An enhancer sequence can be inserted into a vector to enhance transcription of DNA encoding a light chain or heavy chain belonging to an antibody to factor Bb according to the invention by higher eukaryotes. Enhancers are cis-acting DNA elements, typically 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientationally and positionally independent and can be located in the positions of both 5 ′ and 3 ′ to the transcriptional unit. Several enhancer sequences are known that are accessible from mammalian genes (e.g., globin, elastase, albumin, alpha-fetoprotein and insulin). In this case, as a rule, enhancers from viruses are used. Prior art SV40 enhancer, cytomegalovirus early promoter enhancer, polyoma enhancer, and adenovirus enhancer are typical enhancer elements for activating eukaryotic promoters. Although the enhancer can be in the vector as 5 ', and 3' to the coding sequence, as a rule, it is located 5 'from the promoter. To stimulate extracellular secretion of an antibody, a sequence encoding the corresponding native or heterologous sequence (leader sequence or signal peptide) may be included in the expression vector. The choice of signal peptide or leader sequence depends on the type of host cells in which the antibody is to be generated, and a heterologous signal sequence can replace the native signal sequence. Examples of signal peptides that are functional in mammalian host cells include the following: interleukin-7 (IL-7) signal sequence described in US Pat. No. 4,965,195; the interleukin-2 receptor signal sequence described in Cosman et al., 1984, Nature 312: 768; the signal peptide of the interleukin-4 receptor described in patent EP No. 0367566; type I interleukin-1 receptor signal peptide described in US Pat. No. 4,968,607; the signal peptide of the receptor for interleukin-1 type II described in patent EP No. 0460846.

[00219] Expression vectors for the expression of the claimed antibodies according to the invention can be constructed from a source vector, such as a commercially available vector. Such vectors may or may not contain all the necessary flanking sequences. If one or more of the flanking sequences described herein is not initially present in a vector, they can be obtained separately and ligated into a vector. The methods used to obtain each of the flanking sequences are well known to those skilled in the art.

[00220] After the vector has been constructed and the nucleic acid molecule encoding the light chain, heavy chain or light chain and heavy chain constituting the antigen binding sequence for factor Bb has been inserted into the corresponding region of the vector, the entire vector can be introduced into a suitable cell the host for amplification and / or expression of the polypeptide. Transfer of the expression vector for the anti-Bb factor antibody to a selected host cell can be accomplished by well-known methods, including transfection, infection, co-precipitation with calcium phosphate, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known methods. The method chosen will partially depend on the type of host cell used. These methods and other suitable methods are well known to those skilled in the art and are given, for example, in Sambrook et al., 2001, supra.

[00221] When cultured under appropriate conditions, the host cell synthesizes an antibody to factor Bb, which can subsequently be collected from the culture medium (if the host cell secretes into the medium) or directly from the host cell that produces it (if not secretes). The selection of a suitable host cell will depend on various factors, such as the necessary expression levels, polypeptide modifications, desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule. The host cell may be eukaryotic or prokaryotic.

[00222] Mammalian cell lines available as hosts are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary cells ( CHO), HeLa cells, newborn hamster kidney cells (BHK), monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g. Hep G2), and a large number of other cell lines. In certain embodiments of the invention, cell lines can be selected by determining which cell lines have high levels of expression and constitutively produce antibodies with the Bb factor binding property. In another embodiment, a cell line may be selected from a B cell differentiation line that does not produce its own antibody but has the ability to produce and secrete a heterologous antibody.

The use of antibodies to factor Bb for diagnostic and therapeutic purposes

[00223] The antibodies of the invention are useful for detecting factor Bb in biological samples and determining cells or tissues that produce a factor Bb protein. For example, antibodies to factor Bb according to the invention can be used in diagnostic assay methods, for example, binding assays, to detect and / or quantify factor Bb expressed in tissue or cell. Elevated levels of factor Bb may indicate the presence of diseases such as visual impairment, cancer, infection, and / or ulcerative colitis. Reduced levels of Bb factor may indicate cirrhosis, glomerulonephritis, hereditary angioedema, hepatitis, kidney transplant rejection, lupus nephritis, malnutrition and / or systemic lupus erythematosus.

[00224] In some embodiments of the invention, antibodies of the invention that specifically bind to factor Bb can be used in the treatment of factor Bb-mediated diseases in a patient in need thereof. In addition, an antibody to factor Bb according to the invention can be used to inhibit the formation of factor Bb complex with other complement proteins, thereby modulating the biological activity of factor Bb in a cell or tissue. Antibodies that bind to factor Bb can thus modulate and / or block interaction with other binding compounds and, therefore, can have therapeutic use in alleviating factor Bb-mediated diseases.

[00225] In some embodiments, antibodies to factor Bb can block the protease activity of factor Bb. In some cases, the binding of factor Bb to antibodies to factor Bb can lead to disruption of the signal transduction cascade induced by factor Bb.

Diagnostic methods

[00226] The antibodies of the invention can be used for diagnostic purposes to detect, diagnose or monitor diseases and / or pathological conditions associated with factor Bb or factor B. The invention provides for the detection of the presence of factor Bb in a sample using classical immunohistological methods known to those skilled in the art. technical field (e.g., Tijssen, 1993, Practice and Theory of Enzyme Immunoassays, vol 15 (Eds RH Burdon and PH van Knippenberg, Elsevier, Amsterdam); Zola, 1987, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al., 1985, J. Cell. Biol. 101: 976-985; Jalka nen et al., 1987, J. Cell Biol. 105: 3087-3096). Identification of factor Bb can be carried out in vivo or in vitro.

[00227] The diagnostic applications provided herein include the use of antibodies to detect expression of factor Bb and / or binding to factor Bb. Examples of methods used in detecting the presence of factor Bb include immunoassay methods such as enzyme immunosorbent assay (ELISA) and radioimmunoassay (RIA).

[00228] For diagnostic applications, an antibody can typically be labeled with a detectable labeling group. Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g. ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I), fluorescent groups (e.g. , FITC, rhodamine, phosphors based on lanthanide complexes), enzymatic groups (for example, horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups or predetermined polypeptide epitopes recognized by a secondary molar reporter (e.g. , communication sites secondary antibodies, metal binding domains, epitope labels). In some embodiments of the invention, the labeling group is coupled to the antibody via spacer legs of various lengths to reduce potential steric mismatch. Various methods for labeling proteins that can be used to carry out the present invention are known in the art.

[00229] In one aspect of the invention, a method for determining cells or cells that express factor Bb is provided. In a specific embodiment, the antibody is labeled with a labeling group and binding of the labeled antibody to factor Bb is determined. In another specific embodiment, the binding of an antibody to factor Bb can be determined in vivo. In another specific embodiment, the antibody / factor Bb complex is isolated and measured using methods known in the art. See, for example, Harlow and Lane, 1988, Antibodies: A Laboratory Manual, New York: Cold Spring Harbor (ed. 1991 and periodicals); John E. Coligan, ed., 1993, Current Protocols In Immunology New York: John Wiley & Sons.

[00230] In another aspect of the invention, a method for determining the presence of a test molecule that competes with antibodies of the invention for binding to factor Bb is provided. An example of such an analysis involves determining the amount of free antibody in a solution containing a certain amount of factor Bb in the presence or absence of a test molecule. An increase in the amount of free antibody (i.e., an antibody not bound to factor Bb) indicates that the test molecule is able to compete with the antibody for binding to factor Bb. In one embodiment of the invention, the antibody is labeled with a labeling group. Alternatively, a test molecule is labeled and the amount of free test molecule in the presence or absence of an antibody is monitored.

Indications

[00231] The complement system has been shown to be associated with several acute and chronic pathological conditions, including atherosclerosis, ischemia-reperfusion after acute myocardial infarction, nephritis with Shenlein-Genoch purpura, hemorrhagic vasculitis, rheumatoid arthritis, arteritis, aneurysm, stroke, cardiitis, hemorrhagic shock, damage with tissue crushing, multiple organ failure, hypovolemic shock and intestinal ischemia, transplant rejection, cardiac surgery, ChKTA, spontaneous miscarriage, neuronal damage waiting, spinal cord injury, myasthenia gravis, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Guillain-Barré syndrome, Parkinson's disease, Alzheimer's disease, acute respiratory failure syndrome, asthma, chronic obstructive pulmonary disease, acute post-transfusion lung injury, acute lung damage , Goodpasture disease, myocardial infarction, inflammation after applying cardiopulmonary bypass, cardiopulmonary bypass, septic shock, graft rejection, cenotransplantation, burn injuries, systemic lupus erythematosus, membranous nephritis, Berger’s disease, psoriasis, pemphigoid, dermatomyositis, antiphospholipid syndrome, inflammatory bowel disease, hemodialysis, leukopheresis, plasmapheresis, heparin-induced extracorporeal oxigen, .

[00232] Macular degenerative diseases, such as all stages of age-related macular degeneration (AMD), including dry and wet (non-exudative and exudative) forms, choroidal neovascularization (CNV), uveitis, diabetic and other ischemic retinopathy, as well as other intraocular non-ocular diseases such as diabetic macular edema, pathological myopia, von Hippel-Lindau disease, histoplasmosis of the eye, central retinal vein occlusion (OCVS), branched retinal vein occlusion (RVS), neovascular corneal isation and retinal neovascularization. A preferred group of complementary associated pathological conditions of the eye includes age-related macular degeneration (AMD), including non-exudative (wet) and exudative (dry or atrophic) AMD, choroidal neovascularization (CNV), diabetic retinopathy (DR) and endophthalmitis.

[00233] The antibodies against Bb factor disclosed in the present invention can be used in combination with one or more cytokines, lymphokines, hematopoietic factors and / or anti-inflammatory agents.

[00234] The treatment of the diseases and disorders listed herein may include the use of first-line drugs to control pain and inflammation in combination (pre-treatment, subsequent treatment, or simultaneous treatment) with treatment of one or more of the antibodies provided herein. These drugs are classified as non-steroidal anti-inflammatory drugs (NSAIDs). Secondary treatment includes corticosteroids, slow-acting antirheumatic drugs (SAARD), or disease-modifying drugs (DM). Information on the following compounds can be found in The Merck Manual of Diagnosis and Therapy, Sixteenth Edition, Merck, Sharp & Dohme Research Laboratories, Merck & Co., Rahway, N.J. (1992) and in Pharmaprojects, PJB Publications Ltd.

[00235] In a specific embodiment, the present invention relates to the use of an antibody and any one or more NSAIDs for the treatment of the diseases and disorders listed herein. The anti-inflammatory effect of NSAIDs is associated, at least in part, with inhibition of prostaglandin synthesis (Goodman and Gilman in "The Pharmacological Basis of Therapeutics," MacMillan 7th Edition (1985)). NSAIDs can be divided into the following nine groups: (1) salicylic acid derivatives; (2) derivatives of propionic acid; (3) derivatives of acetic acid; (4) phenamic acid derivatives; (5) carboxylic acid derivatives; (6) derivatives of butyric acid; (7) oxicams; (8) pyrazoles; and (9) pyrazolones.

[00236] In another specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more salicylic acid derivatives, prodrug esters or pharmaceutically acceptable salts thereof. Such salicylic acid derivatives, their prodrug esters or pharmaceutically acceptable salts include: acetaminosalol, aloxiprine, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, choline-magnesium trisalicylate, magnesium salicylate, choline salicylate, diflunisal, etosalic acid salicylicate, salosylate, imidazolsalicylate, lysine acetylsalicylate, mesalamine, morpholinsalicylate, 1-naphthyl salicylate, parsalmide, parsalmide, phenylacetylsalicylate, phenyl salicylate, salacetamide, salicylamine, O-acetic acid y, salsalate, sodium salicylate, and sulfasalazine. Structurally related derivatives of salicylic acid having similar analgesic and anti-inflammatory properties are also included in this group.

[00237] In a further specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more propionic acid derivatives, prodrug esters thereof or pharmaceutically acceptable salts thereof. Derivatives of propionic acid, their prodrug esters or pharmaceutically acceptable salts include: alaminoprofen, benoxaprofen, bucloxic acid, caprofen, dexindoprofen, phenoprofen, flunxaprofen, fluprofen, flurbiprofen, furclofrofen, ibuprofenfenfenfenfenfenmenfenfenfenfenfenmenfenfenfenfenfenfenfenfenfenfenfenfenfenfenfenphen , naproxen, naproxen sodium, oxaprozin, picketoprofen, pimeprofen, pirprofen, pranoprofen, proticinic acid, pyridoxyprofen, suprofen, thiaprofenic acid and thioxaprofen. Structurally related propionic acid derivatives having similar analgesic and anti-inflammatory properties are also included in this group.

[00238] In another specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more acetic acid derivatives, prodrug esters thereof or pharmaceutically acceptable salts thereof. Acetic acid derivatives, prodrug esters thereof or pharmaceutically acceptable salts thereof include: acemetacin, aclofenac, amfenac, bufexamac, cinmetacin, clopirac, delmetacin, diclofenac calcium, diclofenac sodium, etodolac, felbinac, phenclofenac, phenclacoflucaclucofencolefenclofencoinclucofenclofenclofenclofencl , ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, methacic acid, oxametacin, oxspinac, pimetacin, proglumethacin, sulindac, talmethacin, thiaramide, thiopinac, tolmetin, sodium tolmetin, zidomethacin and zome iraq. Structurally related derivatives of acetic acid having similar analgesic and anti-inflammatory properties are also included in this group.

[00239] In another specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more phenamic acid derivatives, prodrug esters thereof or pharmaceutically acceptable salts thereof. Fenamic acid derivatives, their prodrug esters or pharmaceutically acceptable salts thereof comprise: enfenaminovuyu acid, etofenamate, flufenamic acid, izoneksin, meclofenamic acid, meclofenamate sodium, medofenaminovuyu acid, mefenamic acid, niflumic acid, talniflumat, terofenamat, tolfenaminovuyu acid and ufenamate. Structurally related derivatives of fenamic acid having similar analgesic and anti-inflammatory properties are also included in this group.

[00240] In a further specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more carboxylic acid derivatives, their prodrug esters or pharmaceutically acceptable salts. Derivatives of the carboxylic acid, their prodrug esters or pharmaceutically acceptable salts that can be used include: clidanac, diflunisal, flufenizal, inoridine, ketorolac and tinoridine. Structurally related carboxylic acid derivatives having similar analgesic and anti-inflammatory properties are also included in this group.

[00241] In another specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more derivatives of butyric acid, their prodrug esters or pharmaceutically acceptable salts. Derivatives of butyric acid, prodrug esters thereof or pharmaceutically acceptable salts thereof include: paperdisone, butibufen, fenbufen and xenbucin. Structurally related derivatives of butyric acid having similar analgesic and anti-inflammatory properties are also included in this group.

[00242] In another specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more oxicams, their prodrug esters or pharmaceutically acceptable salts. Oxycams, their prodrug esters or pharmaceutically acceptable salts include: droxam, enolic, isoxic, piroxicam, sudoxic, tenoxic and 4-hydroxyl-1,2-benzothiazine-1,1-dioxide-4- (N-phenyl) -carboxamide. Structurally related oxycams having similar analgesic and anti-inflammatory properties are also included in this group.

[00243] In another specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more pyrazoles, prodrug esters or pharmaceutically acceptable salts thereof. Pyrazoles, their prodrug esters or pharmaceutically acceptable salts that can be used include diphenamisole and epirisole. Structurally related pyrazoles having similar analgesic and anti-inflammatory properties are also included in this group.

[00244] In a further specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more pyrazolones, prodrug esters or pharmaceutically acceptable salts thereof. Pyrazolones, their prodrug esters or pharmaceutically acceptable salts that can be used include: apazone, azapropazone, benzpiperilone, feprazone, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzon, propylphenazone, ramifenazone, suzibin. Structurally related pyrazolones having similar analgesic and anti-inflammatory properties are also included in this group.

[00245] In another specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more of the following NSAIDs: ε-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4- hydroxybutyric acid, amixetrine, anithrazaphene, anthrafenin, bendazak, bendazak lysinate, benzydamine, beprosin, broperamol, bukolom, bufesolak, cyproquazone, kloksimat, dazidamine, deboxamet, detomidine, diphen diphenpiramide, diphysalamine, ditazole, emorphazone, fanethylisole, mesylate, fenflumisole, flotafenin, flumizole, fopyrtoline, phosphosal, guaimesone, guinzolefinlomisolefin, lonefismolamine, lonefismolamine, lonefinolamine, lonefinflomolamine, lonefismolinfilmeneflinmene, lonefinflomolinfilminefin, lonefismolinflominefin, lonefismolinflominefin, lonefismolinfilmeneflin lominefinmene , nimesulide, orgotein, opranoxin, oxacetrol, oxapadol, paraniline, perisoxal, perisoxal citrate, pifoxime, piproxen, pyrazolac, pirfenidone, proquazone, proxazole, thielavin B , tiflamisole, thimegadine, toctin, tolpadol, tryptamide and preparations designated by the company code 480156S, AA861, AD1590, AFP802, AFP860, AI77B, AP504, AU8001, VRPC, BW540C, CHINOIN 127, CN100, EB506, EL, 508, 5038, EL , GV3658, ITF182, KCNTEI6090, KME4, LA2851, MR714, MR897, MY309, ONO3144, PR823, PV102, PV108, R830, RS2131, SCR152, SH440, SIR133, SPAS510, SQ27239, ST281, 401, TA281, 600600 -benzoyl-1-indanecarboxylic acid), TVX2706, U60257, UR2301 and WY41770. Structurally related NSAIDs having similar analgesic and anti-inflammatory properties with NSAIDs are also included in this group.

[00246] In another specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more corticosteroids, their prodrug esters or pharmaceutically acceptable salts for the treatment of the diseases and disorders listed herein including acute and chronic inflammation, such as rheumatic diseases, graft versus host disease, and multiple sclerosis. Corticosteroids, their prodrug esters or pharmaceutically acceptable salts include hydrocortisone and compounds derived from hydrocortisone, such as 21-acetoxypregnenolone, alklomerazone, algestone, amcinonide, beclomethasone, betamethasone, betamethasone valerate, budenizonide, chlorprednisolone ketone, chloropredone ketone, butyrate, clocortocolon, cloprednol, corticosterone, cortisone, cortivazole, deflazacone, desonide, deoxymerazone, dexamethasone, diflorazon, diflucortolone, difluprednat, enoxolone, fluase ort, flucloronide, flumethasone, flumethasone pivalate, flucinolone acetonide, flunisolide, fluocinonide, fluorotsinolon acetonide, fluorokortin butyl, fluorokortolon, fluorokortolon hexanoate, diflucortolone valerate, fluorometolon, fluperolon acetate, Fluprednidene acetate, fluprednisolone, flurandenolid, formocortal, galtsinonid, halometasone, halopredone acetate , hydrocortamate, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone phosphate, hydrocortisone 21-sodium succinate, hydrocortisone tebutate, mazipredone, medrisone, meprednisone, m tilprednicolone, mometasone furoate, parametasone, predicarbate, prednisolone, prednisolone 21-dihedriaminoacetate, prednisolone sodium phosphate, prednisolone sodium succinate, prednisolone sodium 21-m-sulfobenzoate, prednisolone sodium 21-stearolonate, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisolone, prednisone prednilidene, prednilidene 21-diethylaminoacetate, thixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide and triamcinolone hexacetonide. Structurally related corticosteroids having similar analgesic and anti-inflammatory properties are also included in this group.

[00247] In another specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment, or simultaneous treatment) with any one or more slow-acting anti-rheumatic drugs (SAARD) or disease-modifying anti-rheumatic drugs (DMARD), their prodrug esters or pharmaceutically acceptable salts for the treatment of the diseases and disorders listed herein, including acute and chronic inflammation, as rheumatic disease, a disease "graft versus host" and multiple sclerosis. SAARD or DMARD, prodrug esters or pharmaceutically acceptable salts thereof include: sodium allocupreide, auranofin, aurothioglucose, aurothioglucanide, azathioprine, sodium brequinar, butylamine, calcium 3-aurothio-2-propanol-1-sulfonate, chlorambinocrocyl, , cyclophosphamide, cyclosporine, dapsone, 15-deoxyspergualin, diacerein, glucosamine, gold salts (e.g., cycloquin gold salt, thiomalate gold salt, thiosulfate gold salt), hydroxychloroquine, hydroxychloroquin sulfate, hydroxyurea, kebuzon, lion mizol, lobenzarit, melittin, 6-mercaptopurine, methotrexate, mizoribine, mycophenolate mofetil, marjoram, nitrogen mustard, D-pennitsillamin such pyridinol imidazoles as SKNF86002 and SB203580, rapamycin, thiols, thymopoietin and vincristine. Structurally related SAARD or DMARD having similar analgesic and anti-inflammatory properties are also included in this group.

[00248] In another specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more of COX2 inhibitors, their prodrug esters or pharmaceutically acceptable salts for the treatment of the diseases listed herein and disorders, including acute and chronic inflammation. Examples of COX2 inhibitors, prodrug esters or pharmaceutically acceptable salts thereof include, for example, celecoxib. Structurally related COX2 inhibitors having similar analgesic and anti-inflammatory properties are also included in this group. Examples of COX-2 selective inhibitors include, but are not limited to, etoricoxib, valdecoxib, celecoxib, lycofelone, lumiracoxib, rofecoxib, and the like.

[00249] In another specific embodiment, the present invention relates to the use of an antibody in combination (pretreatment, subsequent treatment or simultaneous treatment) with any one or more antimicrobial agents, their prodrug esters or pharmaceutically acceptable salts for the treatment of the diseases listed herein and disorders, including acute and chronic inflammation. Antimicrobial agents include, for example, a wide class of penicillins, cephalosporins and other beta-lactams, aminoglycosides, azoles, quinolones, macrolides, rifamycins, tetracyclines, sulfonamides, lincosamides and polymyxins. The penicillins include, but are not limited to penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, floksatsillin, ampicillin, ampicillin / sulbactam, amoxicillin, amoxicillin / clavulanate, hetacillin, cyclacillin, vakampitsillin, carbenicillin, carbenicillin indanyl, ticarcillin , ticarcillin / clavulanate, azlocillin, meslocillin, peperacillin and mecillins. Cephalosporins and other beta-lactams include, but are not limited to, cephalotin, cefapirin, cephalexin, cefradine, cefazolin, cefadroxil, cefaclor, cefamandole, cefotetan, cefoxitin, cefroxefoxetoxone cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefoximetse cefroximetse , ceftazidime, imipenem and aztreonam. Aminoglycosides include, but are not limited to, streptomycin, gentamicin, tobramycin, amikacin, netilmicin, kanamycin and neomycin. Azoles include, but are not limited to, fluconazole. Quinolones include, but are not limited to, nalidixic acid, norfloxacin, enoxacin, ciprofloxacin, ofloxacin, sparfloxacin and temafloxacin. Macrolides include, but are not limited to, erythomycin, spiramycin, and azithromycin. Rifamycins include, but are not limited to, rifampin. Tetracyclines include, but are not limited to, spicycline, chlortetracycline, clomocycline, demeclocycline, deoxycycline, guamecycline, lymecycline, meclocycline, metacycline, minocycline, oxytetracycline, penimepicycline, pipacycline, rolitracycline, sanocin, and tetracycline. Sulfonamides include, but are not limited to, sulfonamide, sulfamethoxazole, sulfacetamide, sulfadiazine, sulfisoxazole and co-trimoxazole (trimethoprim / sulfamethoxazole). Lincosamides include, but are not limited to, clindamycin and lincomycin. Polymyxins (polypeptides) include, but are not limited to, polymyxin B and colistin.

Methods of treatment Pharmaceutical preparations, routes of administration

[00250] Disclosed are compositions comprising a therapeutically effective amount of one or a plurality of antibodies of the invention together with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant. In addition, the invention provides methods for treating a patient by administering such a pharmaceutical composition. The patient may be either a human subject or an animal subject.

[00251] Pharmaceutical compositions containing one or more antibodies can be used to reduce the activity of factor Bb. Pharmaceutical compositions containing one or more antibodies can be used to treat the effects, symptoms and / or pathologies associated with the activity of factor Bb. Pharmaceutical compositions containing one or more antibodies can be used in methods of inhibiting the complement pathway and / or binding of factor Bb to other complement proteins. In certain embodiments, the antibody inhibits the protease activity of factor Bb. In further embodiments, pharmaceutical compositions containing one or more antibodies can be used in methods for inhibiting the protease activity of factor Bb. Pharmaceutical compositions containing one or more antibodies can be used in methods of treating the effects, symptoms and / or pathologies associated with the activity of factor Bb. Pharmaceutical compositions containing one or more antibodies can be used in methods for inhibiting MAK production. Pharmaceutical compositions containing one or more antibodies can be used in methods of inhibiting macular degeneration.

[00252] Preferably, acceptable drug materials are non-toxic to recipients at the dosages and concentrations used. In specific embodiments of the invention, pharmaceutical compositions are provided comprising an effective amount of antibodies to Bb factor.

[00253] In certain embodiments of the invention, acceptable materials for the preparations are non-toxic to recipients at the dosages and concentrations used. In certain embodiments of the invention, the pharmaceutical composition may contain materials for preparations for modifying, maintaining or maintaining, for example, pH, osmolarity, viscosity, transparency, color, isotonicity, odor, sterility, stability, rate of dissolution or release, absorption or penetration of the composition. In such embodiments, suitable drug materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrosulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); colorants, flavors and diluents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan ester, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol); means for delivery; thinners; excipients and / or pharmaceutical adjuvants. See, REMINGTON'S PHARMACEUTICAL SCIENCES, 18 ^th Edition, (AR Genrmo, ed.), 1990, Mack Publishing Company.

[00254] In certain embodiments of the invention, the optimal pharmaceutical composition is determined by a person skilled in the art, depending, for example, on the route of administration, the delivery format, and the required dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, above. In certain embodiments of the invention, such compositions may affect the physical condition, stability, in vivo release rate and in vivo elimination rate of antibodies of the invention. In certain embodiments of the invention, a base solution or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature. For example, a suitable base solution or carrier may be water for injection, physiological saline or artificial cerebrospinal fluid, optionally supplemented with other materials common in parenteral compositions. Additional typical stock solutions are buffered saline or saline mixed with serum albumin. In specific embodiments of the invention, the pharmaceutical compositions comprise Tris buffer with a pH of about 7.0-8.5 or acetate buffer with a pH of about 4.0-5.5, and may further comprise sorbitol or a suitable substituent thereof. In certain embodiments of the invention, compositions containing an antibody against Bb factor can be prepared for storage by mixing the selected composition having the desired degree of purification with optional agents (REMINGTON'S PHARMACEUTICAL SCIENCES, above) in the form of a lyophilized tablet or aqueous solution. In addition, in certain embodiments of the invention, the anti-Bb factor antibody product can be prepared as a lyophilisate using appropriate excipients, such as sucrose.

[00255] The pharmaceutical compositions of the invention may be intended for parenteral delivery. Alternatively, the compositions may be intended for inhalation or for delivery through the digestive tract, for example, oral. The preparation of such pharmaceutically acceptable compositions is within the competence of the art.

[00256] The components of the preparation are preferably present in concentrations that are acceptable given the site of administration. In certain embodiments of the invention, buffers are used to maintain the composition at a physiological pH or slightly lower pH, typically in the pH range of about 5 to about 8.

[00257] If parenteral administration is contemplated, the therapeutic compositions for use in the present invention may be in the form of a pyrogen-free, parenterally acceptable aqueous solution containing the necessary antibody to factor Bb in a pharmaceutically acceptable base solution. A particularly suitable parenteral injection stock solution is sterile distilled water, to which the anti-factor Bb antibody is added, in the form of a sterile, isotonic solution with an appropriate preservative. In certain embodiments of the invention, the preparation may include mixing the desired molecule with an agent, such as injectable microspheres, biodegradable particles, polymeric compounds (such as polylactic acid or polyglycolic acid), granules or liposomes, which can provide controlled or prolonged release of the product, which may be delivered by depot injection. In certain embodiments of the invention, it is also possible to use hyaluronic acid, which helps to prolong the circulation period. In certain embodiments of the invention, implantable drug delivery devices may be used to deliver the desired antibody.

[00258] The pharmaceutical compositions of the invention can be prepared for inhalation. In these embodiments, anti-Bb factor antibodies are preferably formulated as a dry, respirable powder. In specific embodiments of the invention, inhalation solutions containing factor Bb can also be prepared with a propellant for aerosol delivery. In certain embodiments of the invention, the solutions can be sprayed. Inhalation administration and methods for preparing preparations for it are further described in International Patent Application No. PCT / US94 / 001875, which is incorporated by reference and describes the inhalation delivery of chemically modified proteins. It is also contemplated that the preparations may be administered orally. Antibodies to factor Bb that are administered in this manner can be prepared with or without carriers commonly used in formulating solid dosage forms, such as tablets and capsules. In certain embodiments of the invention, the capsule can be prepared so as to ensure the release of the active part of the drug at the point of the gastrointestinal tract with maximum bioavailability and minimal presystemic degradation. Additional agents may be included to facilitate absorption of antibodies to Bb factor. It is also possible to use diluents, flavorings, refractory waxes, vegetable oils, lubricants, suspending agents, substances to improve the disintegration of tablets and binders.

[00259] The pharmaceutical compositions of the invention preferably comprise an effective amount of one or more antibodies to factor Bb in admixture with non-toxic adjuvants which are suitable for the manufacture of tablets. By dissolving the tablets in sterile water or another suitable basic solution, a solution in the form of a unit dose can be obtained. Suitable excipients include, but are not limited to, inert diluents such as calcium carbonate, sodium carbonate or bicarbonate, lactose or calcium phosphate; or binding agents, such as starch, gelatin or gum arabic; or lubricants, such as magnesium stearate, stearic acid or talc.

[00260] The existence of additional pharmaceutical compositions is apparent to those skilled in the art, including sustained or controlled delivery of anti-Bb factor antibodies. Methods for preparing various other sustained or controlled delivery agents, such as liposome carriers, biodegradable microparticles or porous granules and depot injections, are also known to those skilled in the art. See, for example, International Patent Application No. PCT / US93 / 00829, which is incorporated by reference and describes the controlled release of porous polymer microparticles for the delivery of pharmaceutical compositions. Sustained-release preparations may include semipermeable polymer matrices in the form of molded articles, for example, films or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides (as disclosed in US Pat. No. 3,773,919 and published European patent application EP 058481, which are incorporated by reference), copolymers of L-glutamic acid and gamma ethyl L-glutamate (Sidman et al., 1983, Biopolymers 2: 547-556), poly (2-hydroxyethyl-inetacrylate) (Langer et al., 1981, J. Biomed. Mater. Res. 15: 167-277 and Langer, 1982, Chem. Tech . 12: 98-105), ethylene vinyl acetate (Langer et al., 1981, supra) or poly-D (-) - 3-hydroxybutyric acid (published European Patent Application No. EP 133988). Sustained release compositions may also include liposomes, which can be made by any of several methods known in the art. See, for example, Eppstein et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82: 3688-3692; published applications for European patent No. EP 036676; EP 088046 and EP 143949, incorporated by reference.

[00261] The pharmaceutical compositions used for in vivo administration are generally provided in the form of sterile preparations. Sterilization can be accomplished by filtration through sterile filter membranes. If the composition is lyophilized, sterilization can be carried out both before and after lyophilization and recovery. Compositions for parenteral administration can be stored in lyophilized form or in solution. Parenteral compositions are generally placed in a container with a sterile inlet, for example, a bag for intravenous solutions or a vial with a stopper pierced by a hypodermic needle for injection.

[00262] After the preparation of the pharmaceutical composition, it can be stored in sterile vials in the form of a solution, suspension, gel, emulsion, solid preparation, crystal, or as an anhydrous or lyophilized powder. Such preparations can be stored both in a ready-to-use form and in a form (for example, lyophilized), which is reconstituted before administration. The invention also provides kits for preparing a unit dose for administration. The kits of the invention may each contain a first container containing dry protein and a second container containing an aqueous solution. In certain embodiments of the invention, kits are also provided containing single and multi-chamber prefilled syringes (e.g., syringes with liquid and syringes with lyophilisate).

[00263] The therapeutically effective amount of a pharmaceutical composition containing an antibody to Bb factor B depends on, for example, the therapeutic context and circumstances. One skilled in the art will recognize that dosage levels suitable for treatment will vary, in part, depending on the molecule delivered, the indications of the antibody against Bb factor, route of administration, and size (body weight, body surface area, or organ size ) and / or condition (age and general health) of the patient. In certain embodiments of the invention, the attending physician may titrate the dosage and modify the route of administration to achieve the optimal therapeutic effect. A typical dosage may correspond to a range of from about 0.1 μg / kg to about 30 mg / kg or more, depending on the above factors. In specific embodiments of the invention, the dosage may correspond to a range from 0.1 μg / kg to about 30 mg / kg, optionally from 1 μg / kg to about 30 mg / kg, or from 10 μg / kg to about 5 mg / kg.

[00264] The dosage often depends on the pharmacokinetic parameters of the particular Bb factor antibody used in the preparation. As a rule, the attending physician administers the composition until a dosage is achieved at which the desired effect is achieved. Therefore, the composition can be administered in a single dose or in two or more doses (which may or may not contain the same amount of the desired molecule) over time or as a long-term infusion by means of an implanted device or catheter. Additional adjustment of the appropriate dosage is usually carried out by specialists in this field of technology and corresponds to the range of tasks they perform. Suitable dosages can be clarified by using appropriate dose-response data. In certain embodiments of the invention, the antibodies of the invention can be administered to patients over an extended period of time. Continuous administration of the antibody of the invention minimizes unwanted immune or allergic responses typically associated with antibodies that are not fully human, for example, an antibody obtained against a human antigen in an organism of a non-human animal, for example, an incompletely human antibody or a non-human antibody obtained in an organism of a species different from a person.

[00265] The route of administration of the pharmaceutical composition corresponds to known methods, for example, oral, by intravenous injection, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intravitreal, subretinal, intraarterial, intraportal or intracranial; using sustained release systems or an implantable device. In certain embodiments of the invention, the compositions may be administered by bolus injection or continuously by infusion or by means of an implantable device.

[00266] Also, the composition can be administered topically by implantation of a membrane, sponge or other suitable material in which the desired molecule is absorbed or encapsulated. In certain embodiments of the invention in which an implantable device is used, said device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can occur through diffusion, a sustained release bolus, or continuous administration. In the case of ophthalmic implants, the implant can be implanted by intraocular injection, intravitreal injection, subretinal injection, suprachoroidal injection, retrobulbar injection, or subtenon injection.

[00267] It may also be necessary to use pharmaceutical compositions containing an ex vivo antibody to factor Bb of the invention. In such cases, cells, tissues or organs that have been removed from the patient’s body are treated with pharmaceutical compositions containing an anti-Bb factor antibody, after which the cells, tissues and / or organs are implanted back to the patient.

[00268] In particular, antibodies to Bb factor can be delivered by implanting certain cells that have been genetically engineered using methods, such as those described herein, for expression and secretion of antibodies to Bb factor. In certain embodiments of the invention, such cells may be animal or human cells and may be autologous, heterologous, or xenogenic. In certain embodiments of the invention, the cells may be immortalized. In other embodiments, in order to reduce the likelihood of an immunological response, cells can be encapsulated to avoid infiltration of surrounding tissues. In further embodiments, the encapsulation materials are typically biocompatible, semipermeable polymer capsules or membranes that allow the release of protein products but prevent cell destruction by the patient’s immune system or other harmful factors from surrounding tissues.

[00269] All references cited in the text of this description are expressly and fully incorporated herein by reference.

EXAMPLES

[00270] The following examples, including experiments and results, are for illustrative purposes only and do not limit the invention.

Example 1 - Analysis of the binding of antibodies against factor Bb in comparison with an antibody against factor B

®, Menlo Park, CA, USA). Анализ проводили при 30°С в 1х буфере ФСБ (Gibco®, ФСБ, рН 7,2). Образцы перемешивали при 1000 об/мин. Перед проведением анализа сенсоры увлажняли в течение 15 минут.[00271] To determine the kinetics of binding of factor Bb (CompTech®) and the human factor B antigen (CompTech®) to a monoclonal antibody against factor Bb, unlabeled biolayer interferometry (BLI) was used. Affinity measurements were performed using Octet QK ^e equipped with biosensor capture tips with anti-human IgG Fc (ANS) (

®, Menlo Park, CA, USA). The analysis was carried out at 30 ° C in 1x FSB buffer (Gibco®, FSB, pH 7.2). Samples were mixed at 1000 rpm. Before analysis, the sensors were moistened for 15 minutes.

[00272] The purified anti-Bb factor antibody was tested for its binding ability to ANS sensory tips. Tips were loaded using 20 μg / ml anti-Bb factor antibodies. The loading lasted 300 s until a capture level between 1.8 and 2 nm was reached. Factor Bb or factor B antigens were prepared for binding analysis by dilution to a concentration of 50 nM in 1x FSB. An association was initiated and monitored for 200 s, after which the tips were transferred to 1x PBS buffer without factor protein (Gibco, PBS, pH 7.2) to track dissociation. Sensory data was obtained during the experiments, processed and analyzed using Octet 7 data analysis software (Forte Bio).

[00273] Antibody factor Bb antibody selectivity was investigated first by comparing the binding association rates for factor Bb and factor B proteins. This assay was performed using Forte Bio® Octet QK ^e system. The binding measured over 200 s in 50 nM protein preparations indicates that the anti-factor Bb antibody specifically binds to factor Bb, and the binding to factor B is significantly lower (Figure 1).

Example 2 - Functional Analysis of a Monoclonal Antibody Against Factor Bb

GVB0) и кроличьи эритроциты. Затем инкубируют реакцию при 37°С в течение 30 минут в шейкере. Добавляют 1,0 мл холодного GVBE. Смешивают и центрифугируют в течение 3 мин приблиз. при 1000×g или выше, чтобы осадить клетки. Переносят 100 мкл супернатанта в 96-луночный планшет и считывают на 412 ни (SoftMax Pro 4.7.1). Данные анализировали при помощи GraphPad Prism 4.[00274] Hemolysis assay - Activation of the alternative pathway (AP) requires higher serum concentrations than the classic pathway. In the general case, a final concentration of 5 mM Mg ++ in the presence of 5 mM EDTA is used for analysis, while EDTA predominantly chelates Ca ⁺⁺ . AP in most mammalian species is activated spontaneously with rabbit erythrocytes, therefore, they are a convenient target. Rabbit erythrocytes (Complement Technology, Inc.) are prepared by washing 3 times with GVB0 (CompTech product) and resuspending at 5 × 10 ⁸ / ml. Different amounts of anti-factor Bb antibody were diluted with GVB0. Mix 100 μl of the reaction on ice from a series of serial dilutions of antibodies against factor Bb, 0.1 M MgEDTU (CompTech product), 1/2 NPP (normal human serum, diluted

GVB0) and rabbit red blood cells. Then the reaction is incubated at 37 ° C for 30 minutes in a shaker. Add 1.0 ml of cold GVBE. Mix and centrifuge for approximately 3 minutes. at 1000 × g or higher to precipitate cells. Transfer 100 μl of the supernatant to a 96-well plate and read on 412 ni (SoftMax Pro 4.7.1). Data was analyzed using GraphPad Prism 4.

[00275] Results - To determine the effectiveness of antibodies against factor Bb, an AP hemolysis and an IC50 nM (the amount of antibody required to inhibit a 50% hemolysis reaction) were analyzed. The data indicate that the IC50 of the anti-factor Bb antibody disclosed in the present invention is about 40 nM, while the IC50 of the anti-factor B antibody is about 100 nM (Figure 2). Therefore, an anti-factor Bb antibody is about ten times more effective than an anti-factor B antibody according to AP hemolysis analysis.

Example 3 - In vivo Efficiency Model

[00276] The humanized H4L4 antibody 99A12 (SEQ ID NOs: 15 and 11, respectively) was examined in a monkey lung injury model. Intravitreal dosing with H4L4 99A12 antibody provided effective blocking of complement deposition in the retina as compared to the control. These data indicate that local delivery of H4L4 antibody 99A12 is effective in an in vivo model relevant to the treatment of people with macular degeneration and other ocular indications.

Claims (14)

1. An antibody to factor Bb containing a heavy chain and a light chain, wherein said antibody binds to factor Bb with greater affinity than factor B and inhibits complement dependent hemolysis, and where: a) the light chain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 8-11, and the heavy chain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 12-15; or b) the light chain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 24-27, and the heavy chain contains an amino acid sequence selected from the group consisting of SEQ ID NO: 28-31. 2. The antibody according to claim 1, which binds factor Bb with CD, less than about 1 nm. 3. The antibody according to claim 1, characterized in that the antibody blocks the formation of a membrane-attacking complex (MAC) in a patient. 4. The antibody according to claim 1, characterized in that the antibody is an antibody containing the variable domain of the light chain and the variable domain of the heavy chain selected from the amino acid sequences of the variable domain of the light chain and heavy chain: SEQ ID NO: 8 / SEQ ID NO: 12; SEQ ID NO: 8 / SEQ ID NO: 13; SEQ ID NO: 8 / SEQ ID NO: 14; SEQ ID NO: 8 / SEQ ID NO: 15; SEQ ID NO: 9 / SEQ ID NO: 12; SEQ ID NO: 9 / SEQ ID NO: 13; SEQ ID NO: 9 / SEQ ID NO: 14; SEQ ID NO: 9 / SEQ ID NO: 15; SEQ ID NO: 10 / SEQ ID NO: 12; SEQ ID NO: 10 / SEQ ID NO: 13; SEQ ID NO: 10 / SEQ ID NO: 14; and SEQ ID NO: 10 / SEQ ID NO: 15; SEQ ID NO: 11 / SEQ ID NO: 12; SEQ ID NO: 11 / SEQ ID NO: 13; SEQ ID NO: 11 / SEQ ID NO: 14; and SEQ ID NO: 11 / SEQ ID NO: 15. 5. The antibody according to claim 1, characterized in that the antibody is an antibody containing the variable domain of the light chain and the variable domain of the heavy chain selected from the amino acid sequences of the variable domain of the light chain and heavy chain: SEQ ID NO: 24 / SEQ ID NO: 28; SEQ ID NO: 24 / SEQ ID NO: 29; SEQ ID NO: 24 / SEQ ID NO: 30; SEQ ID NO: 24 / SEQ ID NO: 31; SEQ ID NO: 25 / SEQ ID NO: 28; SEQ ID NO: 25 / SEQ ID NO: 29; SEQ ID NO: 25 / SEQ ID NO: 30; SEQ ID NO: 25 / SEQ ID NO: 31; SEQ ID NO: 26 / SEQ ID NO: 28; SEQ ID NO: 26 / SEQ ID NO: 29; SEQ ID NO: 26 / SEQ ID NO: 30; and SEQ ID NO: 26 / SEQ ID NO: 31; SEQ ID NO: 27 / SEQ ID NO: 28; SEQ ID NO: 27 / SEQ ID NO: 29; SEQ ID NO: 27 / SEQ ID NO: 30; and SEQ ID NO: 27 / SEQ ID NO: 31. 6. The antibody according to claim 1, characterized in that the antibody contains the amino acid sequence of the variable domain of the light chain of SEQ ID NO: 11 and the amino acid sequence of the variable domain of the heavy chain of SEQ ID NO: 15. 7. The antibody according to claim 1, characterized in that the antibody contains the amino acid sequence of the variable domain of the light chain of SEQ ID NO: 25 and the amino acid sequence of the variable domain of the heavy chain of SEQ ID NO: 30. 8. The antibody according to claim 1, characterized in that the antibody is a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a humanized antibody, a chimeric antibody, a multispecific antibody, or a fragment of these antibodies. 9. The antibody of claim 8, wherein said antibody fragment is a Fab fragment, Fab fragment, F (ab ') ₂ fragment, Fv fragment, diabody or single chain antibody molecule. 10. The antibody according to claim 8, characterized in that said antibody belongs to type IgG1, IgG2, IgG3 or IgG4. 11. A pharmaceutical composition for treating or preventing an eye disease, comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.

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